Corynebacterium glutamicum genes encoding phosphoenolpyruvate: sugar phosphotransferase system proteins

ABSTRACT

Isolated nucleic acid molecules, designated PTS nucleic acid molecules, which encode novel PTS proteins from  Corynebacterium glutamicum  are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PTS nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated PTS proteins, mutated PTS proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from  C. glutamicum  based on genetic engineering of PTS genes in this organism.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/061298, filed Feb. 17, 2005, which is a continuationapplication of U.S. patent application Ser. No. 09/604231, filed Jun.27, 2000, which claims priority to U.S. Provisional Patent ApplicationNo. 60/142,691, filed on Jul. 1, 1999, and also to U.S. ProvisionalPatent Application No. 60/150,310, filed on Aug. 23, 1999. Thisapplication also claims priority to German Patent Application No.:19942095.5, filed on Sep. 3, 1999, and also to German Patent ApplicationNo.: 19942097.1, filed on Sep. 3, 1999. The entire contents of each ofthe foregoing U.S. and German patent applications are incorporatedherein by this reference.

INCORPORATION OF MATERIAL SUBMITTED ON COMPACT DISCS

This application incorporates herein by reference the material containedon the compact discs submitted herewith as part of this application.Specifically, the file “seqlist” (114 KB) contained on each of Copy 1,Copy 2 and the CRF copy of the Sequence Listing is hereby incorporatedherein by reference. This file was created on Jul. 29, 2006.

BACKGROUND OF THE INVENTION

Certain products and by-products of naturally-occurring metabolicprocesses in cells have utility in a wide array of industries, includingthe food, feed, cosmetics, and pharmaceutical industries. Thesemolecules, collectively termed ‘fine chemicals’, include organic acids,both proteinogenic and non-proteinogenic amino acids, nucleotides andnucleosides, lipids and fatty acids, diols, carbohydrates, aromaticcompounds, vitamins and cofactors, and enzymes. Their production is mostconveniently performed through large-scale culture of bacteria developedto produce and secrete large quantities of a particular desiredmolecule. One particularly useful organism for this purpose isCorynebacterium glutamicum, a gram positive, nonpathogenic bacterium.Through strain selection, a number of mutant strains have been developedwhich produce an array of desirable compounds. However, selection ofstrains improved for the production of a particular molecule is atime-consuming and difficult process.

SUMMARY OF THE INVENTION

The invention provides novel bacterial nucleic acid molecules which havea variety of uses. These uses include the identification ofmicroorganisms which can be used to produce fine chemicals, themodulation of fine chemical production in C. glutamicum or relatedbacteria, the typing or identification of C. glutamicum or relatedbacteria, as reference points for mapping the C. glutamicum genome, andas markers for transformation. These novel nucleic acid molecules encodeproteins, referred to herein as phosphoenolpyruvate:sugarphosphotransferase system (PTS) proteins.

C. glutamicum is a gram positive, aerobic bacterium which is commonlyused in industry for the large-scale production of a variety of finechemicals, and also for the degradation of hydrocarbons (such as inpetroleum spills) and for the oxidation of terpenoids. The PTS nucleicacid molecules of the invention, therefore, can be used to identifymicroorganisms which can be used to produce fine chemicals, e.g., byfermentation processes. Modulation of the expression of the PTS nucleicacids of the invention, or modification of the sequence of the PTSnucleic acid molecules of the invention, can be used to modulate theproduction of one or more fine chemicals from a microorganism (e.g., toimprove the yield or production of one or more fine chemicals from aCorynebacterium or Brevibacterium species).

The PTS nucleic acids of the invention may also be used to identify anorganism as being Corynebacterium glutamicum or a close relativethereof, or to identify the presence of C. glutamicum or a relativethereof in a mixed population of microorganisms. The invention providesthe nucleic acid sequences of a number of C. glutamicum genes; byprobing the extracted genomic DNA of a culture of a unique or mixedpopulation of microorganisms under stringent conditions with a probespanning a region of a C. glutamicum gene which is unique to thisorganism, one can ascertain whether this organism is present. AlthoughCorynebacterium glutamicum itself is nonpathogenic, it is related tospecies pathogenic in humans, such as Corynebacterium diphtheriae (thecausative agent of diphtheria); the detection of such organisms is ofsignificant clinical relevance.

The PTS nucleic acid molecules of the invention may also serve asreference points for mapping of the C. glutamicum genome, or of genomesof related organisms. Similarly, these molecules, or variants orportions thereof, may serve as markers for genetically engineeredCorynebacterium or Brevibacterium species.

The PTS proteins encoded by the novel nucleic acid molecules of theinvention are capable of, for example, transporting high-energycarbon-containing molecules such as glucose into C. glutamicum, or ofparticipating in intracellular signal transduction in thismicroorganism. Given the availability of cloning vectors for use inCorynebacterium glutamicum, such as those disclosed in Sinskey et al.,U.S. Pat. No. 4,649,119, and techniques for genetic manipulation of C.glutamicum and the related Brevibacterium species (e.g., lactofermentum)(Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al.,J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J. Gen.Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of theinvention may be utilized in the genetic engineering of this organism tomake it a better or more efficient producer of one or more finechemicals.

The PTS molecules of the invention may be modified such that the yield,production, and/or efficiency of production of one or more finechemicals is improved. For example, by modifying a PTS protein involvedin the uptake of glucose such that it is optimized in activity, thequantity of glucose uptake or the rate at which glucose is translocatedinto the cell may be increased. The breakdown of glucose and othersugars within the cell provides energy that may be used to driveenergetically unfavorable biochemical reactions, such as those involvedin the biosynthesis of fine chemicals. This breakdown also providesintermediate and precursor molecules necessary for the biosynthesis ofcertain fine chemicals, such as amino acids, vitamins and cofactors. Byincreasing the amount of intracellular high-energy carbon moleculesthrough modification of the PTS molecules of the invention, one maytherefore increase both the energy available to perform metabolicpathways necessary for the production of one or more fine chemicals, andalso the intracellular pools of metabolites necessary for suchproduction.

Further, the PTS molecules of the invention may be involved in one ormore intracellular signal transduction pathways which may affect theyields and/or rate of production of one or more fine chemical from C.glutamicum. For example, proteins necessary for the import of one ormore sugars from the extracellular medium (e.g., HPr, Enzyme I, or amember of an Enzyme II complex) are frequently posttranslationallymodified upon the presence of a sufficient quantity of the sugar in thecell, such that they are no longer able to import that sugar. While thisquantity of sugar at which the transport system is shut off may besufficient to sustain the normal functioning of the cell, it may belimiting for the overproduction of the desired fine chemical. Thus, itmay be desirable to modify the PTS proteins of the invention such thatthey are no longer responsive to such negative regulation, therebypermitting greater intracellular concentrations of one or more sugars tobe achieved, and, by extension, more efficient production or greateryields of one or more fine chemicals from organisms containing suchmutant PTS proteins.

This invention provides novel nucleic acid molecules which encodeproteins, referred to herein as phosphoenolpyruvate:sugarphosphotransferase system (PTS) proteins, which are capable of, forexample, participating in the import of high-energy carbon molecules(e.g., glucose, fructose, or sucrose) into C. glutamicum, and/or ofparticipating in one or more C. glutamicum intracellular signaltransduction pathways. Nucleic acid molecules encoding a PTS protein arereferred to herein as PTS nucleic acid molecules. In a preferredembodiment, the PTS protein participates in the import of high-energycarbon molecules (e.g., glucose, fructose, or sucrose) into C.glutamicum, and also may participate in one or more C. glutamicumintracellular signal transduction pathways. Examples of such proteinsinclude those encoded by the genes set forth in Table 1.

Accordingly, one aspect of the invention pertains to isolated nucleicacid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotidesequence encoding a PTS protein or biologically active portions thereof,as well as nucleic acid fragments suitable as primers or hybridizationprobes for the detection or amplification of PTS-encoding nucleic acid(e.g., DNA or mRNA). In particularly preferred embodiments, the isolatednucleic acid molecule comprises one of the nucleotide sequences setforth in Appendix A or the coding region or a complement thereof of oneof these nucleotide sequences. In other particularly preferredembodiments, the isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes to or is at least about50%, preferably at least about 60%, more preferably at least about 70%,80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%,99% or more homologous to a nucleotide sequence set forth in Appendix A,or a portion thereof. In other preferred embodiments, the isolatednucleic acid molecule encodes one of the amino acid sequences set forthin Appendix B. The preferred PTS proteins of the present invention alsopreferably possess at least one of the PTS activities described herein.

In another embodiment, the isolated nucleic acid molecule encodes aprotein or portion thereof wherein the protein or portion thereofincludes an amino acid sequence which is sufficiently homologous to anamino acid sequence of Appendix B, e.g., sufficiently homologous to anamino acid sequence of Appendix B such that the protein or portionthereof maintains a PTS activity. Preferably, the protein or portionthereof encoded by the nucleic acid molecule maintains the ability toparticipate in the import of high-energy carbon molecules (e.g.,glucose, fructose, or sucrose) into C. glutamicum, and/or to participatein one or more C. glutamicum intracellular signal transduction pathways.In one embodiment, the protein encoded by the nucleic acid molecule isat least about 50%, preferably at least about 60%, and more preferablyat least about 70%, 80%, or 90% and most preferably at least about 95%,96%, 97%, 98%, or 99% or more homologous to an amino acid sequence ofAppendix B (e.g., an entire amino acid sequence selected from thosesequences set forth in Appendix B). In another preferred embodiment, theprotein is a full length C. glutamicum protein which is substantiallyhomologous to an entire amino acid sequence of Appendix B (encoded by anopen reading frame shown in Appendix A).

In another preferred embodiment, the isolated nucleic acid molecule isderived from C. glutamicum and encodes a protein (e.g., a PTS fusionprotein) which includes a biologically active domain which is at leastabout 50% or more homologous to one of the amino acid sequences ofAppendix B and is able to participate in the import of high-energycarbon molecules (e.g., glucose, fructose, or sucrose) into C.glutamicum, and/or to participate in one or more C. glutamicumintracellular signal transduction pathways, or possesses one or more ofthe activities set forth in Table 1, and which also includesheterologous nucleic acid sequences encoding a heterologous polypeptideor regulatory regions.

In another embodiment, the isolated nucleic acid molecule is at least 15nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule comprising a nucleotide sequence of Appendix A.Preferably, the isolated nucleic acid molecule corresponds to anaturally-occurring nucleic acid molecule. More preferably, the isolatednucleic acid encodes a naturally-occurring C. glutamicum PTS protein, ora biologically active portion thereof.

Another aspect of the invention pertains to vectors, e.g., recombinantexpression vectors, containing the nucleic acid molecules of theinvention, and host cells into which such vectors have been introduced.In one embodiment, such a host cell is used to produce a PTS protein byculturing the host cell in a suitable medium. The PTS protein can bethen isolated from the medium or the host cell.

Yet another aspect of the invention pertains to a genetically alteredmicroorganism in which a PTS gene has been introduced or altered. In oneembodiment, the genome of the microorganism has been altered by theintroduction of a nucleic acid molecule of the invention encodingwild-type or mutated PTS sequence as a transgene. In another embodiment,an endogenous PTS gene within the genome of the microorganism has beenaltered, e.g., functionally disrupted, by homologous recombination withan altered PTS gene. In another embodiment, an endogenous or introducedPTS gene in a microorganism has been altered by one or more pointmutations, deletions, or inversions, but still encodes a functional PTSprotein. In still another embodiment, one or more of the regulatoryregions (e.g., a promoter, repressor, or inducer) of a PTS gene in amicroorganism has been altered (e.g., by deletion, truncation,inversion, or point mutation) such that the expression of the PTS geneis modulated. In a preferred embodiment, the microorganism belongs tothe genus Corynebacterium or Brevibacterium, with Corynebacteriumglutamicum being particularly preferred. In a preferred embodiment, themicroorganism is also utilized for the production of a desired compound,such as an amino acid, with lysine being particularly preferred.

In another aspect, the invention provides a method of identifying thepresence or activity of Cornyebacterium diphtheriae in a subject. Thismethod includes detection of one or more of the nucleic acid or aminoacid sequences of the invention (e.g., the sequences set forth inAppendix A or Appendix B) in a subject, thereby detecting the presenceor activity of Corynebacterium diphtheriae in the subject.

Still another aspect of the invention pertains to an isolated PTSprotein or a portion, e.g., a biologically active portion, thereof. In apreferred embodiment, the isolated PTS protein or portion thereof canparticipate in the import of high-energy carbon molecules (e.g.,glucose, fructose, or sucrose) into C. glutamicum, and also mayparticipate in one or more C. glutamicum intracellular signaltransduction pathways. In another preferred embodiment, the isolated PTSprotein or portion thereof is sufficiently homologous to an amino acidsequence of Appendix B such that the protein or portion thereofmaintains the ability to participate in the import of high-energy carbonmolecules (e.g., glucose, fructose, or sucrose) into C. glutamicum,and/or to participate in one or more C. glutamicum intracellular signaltransduction pathways.

The invention also provides an isolated preparation of a PTS protein. Inpreferred embodiments, the PTS protein comprises an amino acid sequenceof Appendix B. In another preferred embodiment, the invention pertainsto an isolated full length protein which is substantially homologous toan entire amino acid sequence of Appendix B (encoded by an open readingframe set forth in Appendix A). In yet another embodiment, the proteinis at least about 50%, preferably at least about 60%, and morepreferably at least about 70%, 80%, or 90%, and most preferably at leastabout 95%, 96%, 97%, 98%, or 99% or more homologous to an entire aminoacid sequence of Appendix B. In other embodiments, the isolated PTSprotein comprises an amino acid sequence which is at least about 50% ormore homologous to one of the amino acid sequences of Appendix B and isable to participate in the import of high-energy carbon molecules (e.g.,glucose, fructose, or sucrose) into C. glutamicum, and/or to participatein one or more C. glutamicum intracellular signal transduction pathways,or has one or more of the activities set forth in Table 1.

Alternatively, the isolated PTS protein can comprise an amino acidsequence which is encoded by a nucleotide sequence which hybridizes,e.g., hybridizes under stringent conditions, or is at least about 50%,preferably at least about 60%, more preferably at least about 70%, 80%,or 90%, and even more preferably at least about 95%, 96%, 97%, 98,%, or99% or more homologous, to a nucleotide sequence of Appendix B. It isalso preferred that the preferred forms of PTS proteins also have one ormore of the PTS bioactivities described herein.

The PTS polypeptide, or a biologically active portion thereof, can beoperatively linked to a non-PTS polypeptide to form a fusion protein. Inpreferred embodiments, this fusion protein has an activity which differsfrom that of the PTS protein alone. In other preferred embodiments, thisfusion protein results in increased yields, production, and/orefficiency of production of a desired fine chemical from C. glutamicum.In particularly preferred embodiments, integration of this fusionprotein into a host cell modulates the production of a desired compoundfrom the cell.

In another aspect, the invention provides methods for screeningmolecules which modulate the activity of a PTS protein, either byinteracting with the protein itself or a substrate or binding partner ofthe PTS protein, or by modulating the transcription or translation of aPTS nucleic acid molecule of the invention.

Another aspect of the invention pertains to a method for producing afine chemical. This method involves the culturing of a cell containing avector directing the expression of a PTS nucleic acid molecule of theinvention, such that a fine chemical is produced. In a preferredembodiment, this method further includes the step of obtaining a cellcontaining such a vector, in which a cell is transfected with a vectordirecting the expression of a PTS nucleic acid. In another preferredembodiment, this method further includes the step of recovering the finechemical from the culture. In a particularly preferred embodiment, thecell is from the genus Corynebacterium or Brevibacterium, or is selectedfrom those strains set forth in Table 3.

Another aspect of the invention pertains to methods for modulatingproduction of a molecule from a microorganism. Such methods includecontacting the cell with an agent which modulates PTS protein activityor PTS nucleic acid expression such that a cell associated activity isaltered relative to this same activity in the absence of the agent. In apreferred embodiment, the cell is modulated for the uptake of one ormore sugars, such that the yields or rate of production of a desiredfine chemical by this microorganism is improved. The agent whichmodulates PTS protein activity can be an agent which stimulates PTSprotein activity or PTS nucleic acid expression. Examples of agentswhich stimulate PTS protein activity or PTS nucleic acid expressioninclude small molecules, active PTS proteins, and nucleic acids encodingPTS proteins that have been introduced into the cell. Examples of agentswhich inhibit PTS activity or expression include small molecules, andantisense PTS nucleic acid molecules.

Another aspect of the invention pertains to methods for modulatingyields of a desired compound from a cell, involving the introduction ofa wild-type or mutant PTS gene into a cell, either maintained on aseparate plasmid or integrated into the genome of the host cell. Ifintegrated into the genome, such integration can random, or it can takeplace by homologous recombination such that the native gene is replacedby the introduced copy, causing the production of the desired compoundfrom the cell to be modulated. In a preferred embodiment, said yieldsare increased. In another preferred embodiment, said chemical is a finechemical. In a particularly preferred embodiment, said fine chemical isan amino acid. In especially preferred embodiments, said amino acid isL-lysine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides PTS nucleic acid and protein moleculeswhich are involved in the uptake of high-energy carbon molecules (e.g.,sucrose, fructose, or glucose) into C. glutamicum, and may alsoparticipate in intracellular signal transduction pathways in thismicroorganism. The molecules of the invention may be utilized in themodulation of production of fine chemicals from microorganisms. Suchmodulation may be due to increased intracellular levels of high-energymolecules needed to produce, e.g, ATP, GTP and other molecules utilizedto drive energetically unfavorable biochemical reactions in the cell,such as the biosynthesis of a fine chemical. This modulation of finechemical production may also be due to the fact that the breakdownproducts of many sugars serve as intermediates or precursors for otherbiosynthetic pathways, including those of certain fine chemicals.Further, PTS proteins are known to participate in certain intracellularsignal transduction pathways which may have regulatory activity for oneor more fine chemical metabolic pathways; by manipulating these PTSproteins, one may thereby activate a fine chemical biosynthetic pathwaysor repress a fine chemical degradation pathway. Aspects of the inventionare further explicated below.

I. Fine Chemicals

The term ‘fine chemical’ is art-recognized and includes moleculesproduced by an organism which have applications in various industries,such as, but not limited to, the pharmaceutical, agriculture, andcosmetics industries. Such compounds include organic acids, such astartaric acid, itaconic acid, and diaminopimelic acid, bothproteinogenic and non-proteinogenic amino acids, purine and pyrimidinebases, nucleosides, and nucleotides (as described e.g in Kuninaka, A.(1996) Nucleotides and related compounds, p. 561-612, in Biotechnologyvol. 6, Rehm et al., eds. VCH: Weinheim, and references containedtherein), lipids, both saturated and unsaturated fatty acids (e.g.,arachidonic acid), diols (e.g., propane diol, and butane diol),carbohydrates (e.g., hyaluronic acid and trehalose), aromatic compounds(e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors(as described in Ullmann's Encyclopedia of Industrial Chemistry, vol.A27, “Vitamins”, p. 443-613 (1996) VCH: Weinheim and references therein;and Ong, A. S., Niki, E. & Packer, L. (1995) “Nutrition, Lipids, Health,and Disease” Proceedings of the UNESCO/Confederation of Scientific andTechnological Associations in Malaysia, and the Society for Free RadicalResearch—Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press,(1995)), enzymes, polyketides (Cane et al. (1998) Science 282: 63-68),and all other chemicals described in Gutcho (1983) Chemicals byFermentation, Noyes Data Corporation, ISBN: 0818805086 and referencestherein. The metabolism and uses of certain of these fine chemicals arefurther explicated below.

A. Amino Acid Metabolism and Uses

Amino acids comprise the basic structural units of all proteins, and assuch are essential for normal cellular functioning in all organisms. Theterm “amino acid” is art-recognized. The proteinogenic amino acids, ofwhich there are 20 species, serve as structural units for proteins, inwhich they are linked by peptide bonds, while the nonproteinogenic aminoacids (hundreds of which are known) are not normally found in proteins(see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97VCH: Weinheim (1985)). Amino acids may be in the D- or L-opticalconfiguration, though L-amino acids are generally the only type found innaturally-occurring proteins. Biosynthetic and degradative pathways ofeach of the 20 proteinogenic amino acids have been well characterized inboth prokaryotic and eukaryotic cells (see, for example, Stryer, L.Biochemistry, 3^(rd) edition, pages 578-590 (1988)). The ‘essential’amino acids (histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan, and valine), so named because theyare generally a nutritional requirement due to the complexity of theirbiosyntheses, are readily converted by simple biosynthetic pathways tothe remaining 11 ‘nonessential’ amino acids (alanine, arginine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline,serine, and tyrosine). Higher animals do retain the ability tosynthesize some of these amino acids, but the essential amino acids mustbe supplied from the diet in order for normal protein synthesis tooccur.

Aside from their function in protein biosynthesis, these amino acids areinteresting chemicals in their own right, and many have been found tohave various applications in the food, feed, chemical, cosmetics,agriculture, and pharmaceutical industries. Lysine is an important aminoacid in the nutrition not only of humans, but also of monogastricanimals such as poultry and swine. Glutamate is most commonly used as aflavor additive (mono-sodium glutamate, MSG) and is widely usedthroughout the food industry, as are aspartate, phenylalanine, glycine,and cysteine. Glycine, L-methionine and tryptophan are all utilized inthe pharmaceutical industry. Glutamine, valine, leucine, iso leucine,histidine, arginine, pro line, serine and alanine are of use in both thepharmaceutical and cosmetics industries. Threonine, tryptophan, andD/L-methionine are common feed additives. (Leuchtenberger, W. (1996)Amino aids—technical production and use, p. 466-502 in Rehm et al.(eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally,these amino acids have been found to be useful as precursors for thesynthesis of synthetic amino acids and proteins, such asN-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan,and others described in Ulmann's Encyclopedia of Industrial Chemistry,vol. A2, p. 57-97, VCH: Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms capable ofproducing them, such as bacteria, has been well characterized (forreview of bacterial amino acid biosynthesis and regulation thereof, seeUmbarger, H. E.(1978) Ann. Rev. Biochem. 47: 533-606). Glutamate issynthesized by the reductive amination of α-ketoglutarate, anintermediate in the citric acid cycle. Glutamine, proline, and arginineare each subsequently produced from glutamate. The biosynthesis ofserine is a three-step process beginning with 3-phosphoglycerate (anintermediate in glycolysis), and resulting in this amino acid afteroxidation, transamination, and hydrolysis steps. Both cysteine andglycine are produced from serine; the former by the condensation ofhomocysteine with serine, and the latter by the transferal of theside-chain β-carbon atom to tetrahydrofolate, in a reaction catalyzed byserine transhydroxymethylase. Phenylalanine, and tyrosine aresynthesized from the glycolytic and pentose phosphate pathway precursorserythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosyntheticpathway that differ only at the final two steps after synthesis ofprephenate. Tryptophan is also produced from these two initialmolecules, but its synthesis is an 11-step pathway. Tyrosine may also besynthesized from phenylalanine, in a reaction catalyzed by phenylalaninehydroxylase. Alanine, valine, and leucine are all biosynthetic productsof pyruvate, the final product of glycolysis. Aspartate is formed fromoxaloacetate, an intermediate of the citric acid cycle. Asparagine,methionine, threonine, and lysine are each produced by the conversion ofaspartate. Isoleucine is formed from threonine. A complex 9-step pathwayresults in the production of histidine from5-phosphoribosyl-1-pyrophosphate, an activated sugar.

Amino acids in excess of the protein synthesis needs of the cell cannotbe stored, and are instead degraded to provide intermediates for themajor metabolic pathways of the cell (for review see Stryer, L.Biochemistry 3^(rd) ed. Ch. 21 “Amino Acid Degradation and the UreaCycle” p. 495-516 (1988)). Although the cell is able to convert unwantedamino acids into useful metabolic intermediates, amino acid productionis costly in terms of energy, precursor molecules, and the enzymesnecessary to synthesize them. Thus it is not surprising that amino acidbiosynthesis is regulated by feedback inhibition, in which the presenceof a particular amino acid serves to slow or entirely stop its ownproduction (for overview of feedback mechanisms in amino acidbiosynthetic pathways, see Stryer, L. Biochemistry, 3^(rd) ed. Ch. 24:“Biosynthesis of Amino Acids and Heme” p. 575-600 (1988)). Thus, theoutput of any particular amino acid is limited by the amount of thatamino acid present in the cell.

B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses

Vitamins, cofactors, and nutraceuticals comprise another group ofmolecules which the higher animals have lost the ability to synthesizeand so must ingest, although they are readily synthesized by otherorganisms, such as bacteria. These molecules are either bioactivesubstances themselves, or are precursors of biologically activesubstances which may serve as electron carriers or intermediates in avariety of metabolic pathways. Aside from their nutritive value, thesecompounds also have significant industrial value as coloring agents,antioxidants, and catalysts or other processing aids. (For an overviewof the structure, activity, and industrial applications of thesecompounds, see, for example, Ullman's Encyclopedia of IndustrialChemistry, “Vitamins” vol. A27, p. 443-613, VCH: Weinheim, 1996.) Theterm “vitamin” is art-recognized, and includes nutrients which arerequired by an organism for normal functioning, but which that organismcannot synthesize by itself. The group of vitamins may encompasscofactors and nutraceutical compounds. The language “cofactor” includesnonproteinaceous compounds required for a normal enzymatic activity tooccur. Such compounds may be organic or inorganic; the cofactormolecules of the invention are preferably organic. The term“nutraceutical” includes dietary supplements having health benefits inplants and animals, particularly humans. Examples of such molecules arevitamins, antioxidants, and also certain lipids (e.g., polyunsaturatedfatty acids).

The biosynthesis of these molecules in organisms capable of producingthem, such as bacteria, has been largely characterized (Ullman'sEncyclopedia of Industrial Chemistry, “Vitamins” vol. A27, p. 443-613,VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley & Sons; Ong, A. S., Niki,E. & Packer, L. (1995) “Nutrition, Lipids, Health, and Disease”Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia, and the Society for Free RadicalResearch—Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press:Champaign, Ill. X, 374 S).

Thiamin (vitamin B₁) is produced by the chemical coupling of pyrimidineand thiazole moieties. Riboflavin (vitamin B₂) is synthesized fromguanosine-5′-triphosphate (GTP) and ribose-5′-phosphate. Riboflavin, inturn, is utilized for the synthesis of flavin mononucleotide (FMN) andflavin adenine dinucleotide (FAD). The family of compounds collectivelytermed ‘vitamin B₆’ (e.g., pyridoxine, pyridoxamine,pyridoxa-5′-phosphate, and the commercially used pyridoxinhydrochloride) are all derivatives of the common structural unit,5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid,(R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-β-alanine) can beproduced either by chemical synthesis or by fermentation. The finalsteps in pantothenate biosynthesis consist of the ATP-drivencondensation of β-alanine and pantoic acid. The enzymes responsible forthe biosynthesis steps for the conversion to pantoic acid, to β-alanineand for the condensation to panthotenic acid are known. Themetabolically active form of pantothenate is Coenzyme A, for which thebiosynthesis proceeds in 5 enzymatic steps. Pantothenate,pyridoxal-5′-phosphate, cysteine and ATP are the precursors of CoenzymeA. These enzymes not only catalyze the formation of panthothante, butalso the production of (R)-pantoic acid, (R)-pantolacton, (R)-panthenol(provitamin B₅), pantetheine (and its derivatives) and coenzyme A.

Biotin biosynthesis from the precursor molecule pimeloyl-CoA inmicroorganisms has been studied in detail and several of the genesinvolved have been identified. Many of the corresponding proteins havebeen found to also be involved in Fe-cluster synthesis and are membersof the nifS class of proteins. Lipoic acid is derived from octanoicacid, and serves as a coenzyme in energy metabolism, where it becomespart of the pyruvate dehydrogenase complex and the α-ketoglutaratedehydrogenase complex. The folates are a group of substances which areall derivatives of folic acid, which is turn is derived from L-glutamicacid, p-amino-benzoic acid and 6-methylpterin. The biosynthesis of folicacid and its derivatives, starting from the metabolism intermediatesguanosine-5′-triphosphate (GTP), L-glutamic acid and p-amino-benzoicacid has been studied in detail in certain microorganisms.

Corrinoids (such as the cobalamines and particularly vitamin B₁₂) andporphyrines belong to a group of chemicals characterized by atetrapyrole ring system. The biosynthesis of vitamin B₁₂ is sufficientlycomplex that it has not yet been completely characterized, but many ofthe enzymes and substrates involved are now known. Nicotinic acid(nicotinate), and nicotinamide are pyridine derivatives which are alsotermed ‘niacin’. Niacin is the precursor of the important coenzymes NAD(nicotinamide adenine dinucleotide) and NADP (nicotinamide adeninedinucleotide phosphate) and their reduced forms.

The large-scale production of these compounds has largely relied oncell-free chemical syntheses, though some of these chemicals have alsobeen produced by large-scale culture of microorganisms, such asriboflavin, Vitamin B₆, pantothenate, and biotin. Only Vitamin B₁₂ isproduced solely by fermentation, due to the complexity of its synthesis.In vitro methodologies require significant inputs of materials and time,often at great cost.

C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Purine and pyrimidine metabolism genes and their corresponding proteinsare important targets for the therapy of tumor diseases and viralinfections. The language “purine” or “pyrimidine” includes thenitrogenous bases which are constituents of nucleic acids, co-enzymes,and nucleotides. The term “nucleotide” includes the basic structuralunits of nucleic acid molecules, which are comprised of a nitrogenousbase, a pentose sugar (in the case of RNA, the sugar is ribose; in thecase of DNA, the sugar is D-deoxyribose), and phosphoric acid. Thelanguage “nucleoside” includes molecules which serve as precursors tonucleotides, but which are lacking the phosphoric acid moiety thatnucleotides possess. By inhibiting the biosynthesis of these molecules,or their mobilization to form nucleic acid molecules, it is possible toinhibit RNA and DNA synthesis; by inhibiting this activity in a fashiontargeted to cancerous cells, the ability of tumor cells to divide andreplicate may be inhibited. Additionally, there are nucleotides which donot form nucleic acid molecules, but rather serve as energy stores(i.e., AMP) or as coenzymes (i.e., FAD and NAD).

Several publications have described the use of these chemicals for thesemedical indications, by influencing purine and/or pyrimidine metabolism(e.g. Christopherson, R. I. and Lyons, S. D. (1990) “Potent inhibitorsof de novo pyrimidine and purine biosynthesis as chemotherapeuticagents.” Med Res. Reviews 10: 505-548). Studies of enzymes involved inpurine and pyrimidine metabolism have been focused on the development ofnew drugs which can be used, for example, as immunosuppressants oranti-proliferants (Smith, J. L., (1995) “Enzymes in nucleotidesynthesis.” Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem Soc.Transact. 23: 877-902). However, purine and pyrimidine bases,nucleosides and nucleotides have other utilities: as intermediates inthe biosynthesis of several fine chemicals (e.g., thiamine,S-adenosyl-methionine, folates, or riboflavin), as energy carriers forthe cell (e.g., ATP or GTP), and for chemicals themselves, commonly usedas flavor enhancers (e.g., IMP or GMP) or for several medicinalapplications (see, for example, Kuninaka, A. (1996) Nucleotides andRelated Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH:Weinheim, p. 561-612). Also, enzymes involved in purine, pyrimidine,nucleoside, or nucleotide metabolism are increasingly serving as targetsagainst which chemicals for crop protection, including fungicides,herbicides and insecticides, are developed.

The metabolism of these compounds in bacteria has been characterized(for reviews see, for example, Zalkin, H. and Dixon, J. E. (1992) “denovo purine nucleotide biosynthesis”, in: Progress in Nucleic AcidResearch and Molecular Biology, vol. 42, Academic Press:, p. 259-287;and Michal, G. (1999) “Nucleotides and Nucleosides”, Chapter 8 in:Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,Wiley: New York). Purine metabolism has been the subject of intensiveresearch, and is essential to the normal functioning of the cell.Impaired purine metabolism in higher animals can cause severe disease,such as gout. Purine nucleotides are synthesized fromribose-5-phosphate, in a series of steps through the intermediatecompound inosine-5′-phosphate (IMP), resulting in the production ofguanosine-5′-monophosphate (GMP) or adenosine-5′-monophosphate (AMP),from which the triphosphate forms utilized as nucleotides are readilyformed. These compounds are also utilized as energy stores, so theirdegradation provides energy for many different biochemical processes inthe cell. Pyrimidine biosynthesis proceeds by the formation ofuridine-5′-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, isconverted to cytidine-5′-triphosphate (CTP). The deoxy-forms of all ofthese nucleotides are produced in a one step reduction reaction from thediphosphate ribose form of the nucleotide to the diphosphate deoxyriboseform of the nucleotide. Upon phosphorylation, these molecules are ableto participate in DNA synthesis.

D. Trehalose Metabolism and Uses

Trehalose consists of two glucose molecules, bound in α,α-1,1 linkage.It is commonly used in the food industry as a sweetener, an additive fordried or frozen foods, and in beverages. However, it also hasapplications in the pharmaceutical, cosmetics and biotechnologyindustries (see, for example, Nishimoto et al., (1998) U.S. Pat. No.5,759,610; Singer, M. A. and Lindquist, S. (1998) Trends Biotech. 16:460-467; Paiva, C. L. A. and Panek, A. D. (1996) Biotech. Ann. Rev. 2:293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose isproduced by enzymes from many microorganisms and is naturally releasedinto the surrounding medium, from which it can be collected usingmethods known in the art.

II. The Phosphoenolpyruvate:Sugar Phosphotransferase System

The ability of cells to grow and divide rapidly in culture is to a greatdegree dependent on the extent to which the cells are able to take upand utilize high energy molecules, such as glucose and other sugars.Different transporter proteins exist to transport different carbonsources into the cell. There are transport proteins for sugars, such asglucose, fructose, mannose, galactose, ribose, sorbose, ribulose,lactose, maltose, sucrose, or raffinose, and also transport proteins forstarch or cellulose degradation products. Other transport systems serveto import alcohols (e.g., methanol or ethanol), alkanes, fatty acids andorganic acids like acetic acid or lactic acid. In bacteria, sugars maybe transported into the cell across the cellular membrane by a varietyof mechanisms. Aside from the symport of sugars with protons, one of themost commonly utilized processes for sugar uptake is the bacterialphosphoenolpyruvate: sugar phosphotransferase system (PTS). This systemnot only catalyzes the translocation (with concomitant phosphorylation)of sugars and hexitols, but it also regulates cellular metabolism inresponse to the availability of carbohydrates. Such PTS systems areubiquitous in bacteria but do not occur in archaebacteria or eukaryotes.

Functionally, the PTS system consists of two cytoplasmic proteins,Enzyme I and HPr, and a variable number of sugar-specific integral andperipheral membrane transport complexes (each termed ‘Enzyme II’ with asugar-specific subscript, e.g., ‘Enzyme II^(Glu)’ for the Enzyme IIcomplex which binds glucose). Enzymes II specific for mono-, di-, oroligosaccharides, like glucose, fructose, mannose, galactose, ribose,sorbose, ribulose, lactose, maltose, sucrose, raffinose, and others areknown. Enzyme I transfers phosphoryl groups from phosphoenolpyruvate(PEP) to the phosphoryl carrier protein, HPr. HPr then transfers thephosphoryl groups to the different Enzyme II transport complexes. Whilethe amino acid sequences of Enzyme I and HPr are quite similar in allbacteria, the sequences for PTS transporters can be grouped intostructurally unrelated families. Further, the number and homologybetween these genes vary from bacteria to bacteria. The E. coli genomeencodes 38 different PTS proteins, 33 of which are subunits belonging to22 different transporters. The M. genitalium genome contains one geneeach for Enzyme I and HPr, and only two genes for PTS transporters. Thegenomes of T. palladium and C. trachomatis contain genes for Enzyme I-and HPr-like proteins but no PTS transporters.

All PTS transporters consist of three functional units, IIA, IIB, andIIC, which occur either as protein subunits in a complex (e.g.,IIA^(Glc)IICB^(Glc)) or as domains of a single polypeptide chain (e.g.,IICBA^(GlcNAc)). IIA and IIB sequentially transfer phosphoryl groupsfrom HPr to the transported sugars. IIC contains the sugar binding site,and spans the inner membrane six or eight times. Sugar translocation iscoupled to the transient phosphorylation of the IIB domain. Enzyme I,HPr, and IIA are phosphorylated at histidine residues, while IIBsubunits are phosphorylated at either cysteine or histidine residues,depending on the particular transporter involved. Phosphorylation of thesugar being imported has the advantage of blocking the diffusion of thesugar back through the cellular membrane to the extracellular medium,since the charged phosphate group cannot readily traverse thehydrophobic core of the membrane.

Some PTS proteins play a role in intracellular signal transduction inaddition to their function in the active transport of sugars. Thesesubunits regulate their targets either allosterically, or byphosphorylation. Their regulatory activity varies with the degree oftheir phosphorylation (i.e., the ratio of the non-phosphorylated to thephosphorylated form), which in turn varies with the ratio ofsugar-dependent dephosphorylation and phosphoenolpyruvate-dependentrephosphorylation. Examples of such intracellular regulation by PTSproteins in E. coli include the inhibition of glycerol kinase bydephosphorylated IIA^(Glc), and the activation of adenylate cyclase bythe phosphorylated version of this protein. Also, the HPr and the IIBdomains of some transporters in these microorganisms regulate geneexpression by reversible phosphorylation of transcriptionantiterminators. In gram-positive bacteria, the activity of HPr ismodulated by HPr-specific serine kinases and phosphatases. For example,HPr phosphorylated at serine-46 functions as a co-repressor of thetranscriptional repressor CcpA. Lastly, it has been found thatunphosphorylated Enzyme I inhibits the sensor kinase CheA of thebacterial chemotaxis machinery, providing a direct link between thesugar binding and transport systems of the bacterium and those systemsgoverning movement of the bacterium (Sonenshein, A. L., et al., eds.Bacillus subtilis and other gram-positive bacteria. ASM: Washington,D.C.; Neidhardt, F. C., et al., eds. (1996) Escherichia coli andSalmonella. ASM Press: Washington, D.C.; Lengeler et al., (1999).Biology of Prokaryotes. Section II, pp. 68-87, Thieme Verlag:Stuttgart).

III. Elements and Methods of the Invention

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as PTS nucleic acid and proteinmolecules, which participate in the uptake of high-energy carbonmolecules (e.g., glucose, sucrose, and fructose) into C. glutamicum, andmay also participate in one or more intracellular signal transductionpathways in these microorganisms. In one embodiment, the PTS moleculesfunction to import high-energy carbon molecules into the cell, where theenergy produced by their degradation may be utilized to power lessenergetically favorable biochemical reactions, and their degradationproducts may serve as intermediates and precursors for a number of othermetabolic pathways. In another embodiment, the PTS molecules mayparticipate in one or more intracellular signal transduction pathways,wherein the presence of a modified form of a PTS molecule (e.g., aphosphorylated PTS protein) may participate in a signal transductioncascade which regulates one or more cellular processes. In a preferredembodiment, the activity of the PTS molecules of the present inventionhas an impact on the production of a desired fine chemical by thisorganism. In a particularly preferred embodiment, the PTS molecules ofthe invention are modulated in activity, such that the yield, productionor efficiency of production of one or more fine chemicals from C.glutamicum is also modulated.

The language, “PTS protein” or “PTS polypeptide” includes proteins whichparticipate in the uptake of one or more high-energy carbon compounds(e.g., mono-, di, or oligosaccharides, such as fructose, mannose,sucrose, glucose, raffinose, galactose, ribose, lactose, maltose, andribulose) from the extracellular medium to the interior of the cell.Such PTS proteins may also participate in one or more intracellularsignal transduction pathways, such as, but not limited to, thosegoverning the uptake of different sugars into the cell. Examples of PTSproteins include those encoded by the PTS genes set forth in Table 1 andAppendix A. For general references pertaining to the PTS system, see:Stryer, L. (1988) Biochemistry. Chapter 37: “Membrane Transport”, W. H.Freeman: New York, p. 959-961; Darnell, J. et al. (1990) Molecular CellBiology Scientific American Books: New York, p. 552-553, and Michal, G.,ed. (1999) Biochemical Pathways: An Atlas of Biochemistry and MolecularBiology, Chapter 15 “Special Bacterial Metabolism”. The terms “PTS gene”or “PTS nucleic acid sequence” include nucleic acid sequences encoding aPTS protein, which consist of a coding region and also correspondinguntranslated 5′ and 3′ sequence regions. Examples of PTS genes includethose set forth in Table 1. The terms “production” or “productivity” areart; recognized and include the concentration of the fermentationproduct (for example, the desired fine chemical) formed within a giventime and a given fermentation volume (e.g., kg product per hour perliter). The term “efficiency of production” includes the time requiredfor a particular level of production to be achieved (for example, howlong it takes for the cell to attain a particular rate of output of afine chemical). The term “yield” or “product/carbon yield” isart-recognized and includes the efficiency of the conversion of thecarbon source into the product (i.e., fine chemical). This is generallywritten as, for example, kg product per kg carbon source. By increasingthe yield or production of the compound, the quantity of recoveredmolecules, or of useful recovered molecules of that compound in a givenamount of culture over a given amount of time is increased. The terms“biosynthesis” or a “biosynthetic pathway” are art-recognized andinclude the synthesis of a compound, preferably an organic compound, bya cell from intermediate compounds in what may be a multistep and highlyregulated process. The terms “degradation” or a “degradation pathway”are art-recognized and include the breakdown of a compound, preferablyan organic compound, by a cell to degradation products (generallyspeaking, smaller or less complex molecules) in what may be a multistepand highly regulated process. The language “metabolism” isart-recognized and includes the totality of the biochemical reactionsthat take place in an organism. The metabolism of a particular compound,then, (e.g., the metabolism of an amino acid such as glycine) comprisesthe overall biosynthetic, modification, and degradation pathways in thecell related to this compound. The language “transport” or “import” isart-recognized and includes the facilitated movement of one or moremolecules across a cellular membrane through which the molecule wouldotherwise be unable to pass.

In another embodiment, the PTS molecules of the invention are capable ofmodulating the production of a desired molecule, such as a finechemical, in a microorganism such as C. glutamicum. Using recombinantgenetic techniques, one or more of the PTS proteins of the invention maybe manipulated such that its function is modulated. For example, aprotein involved in the PTS-mediated import of glucose may be alteredsuch that it is optimized in activity, and the PTS system for theimportation of glucose may thus be able to translocate increased amountsof glucose into the cell. Since glucose molecules are utilized not onlyfor energy to drive energetically unfavorable biochemical reactions,such as fine chemical biosyntheses, but also as precursors andintermediates in a number of fine chemical biosynthetic pathways (e.g.,serine is synthesized from 3-phosphoglycerate). In each case, theoverall yield or rate of production of one of these desired finechemicals may be increased, either by increasing the energy availablefor such production to occur, or by increasing the availability ofcompounds necessary for such production to take place.

Further, many PTS proteins are known to play key roles in intracellularsignal transduction pathways which regulate cellular metabolism andsugar uptake in keeping with the availability of carbon sources. Forexample, it is known that an increased intracellular level of fructose1,6-bisphosphate (a compound produced during glycolysis) results in thephosphorylation of a serine residue on HPr which prevents this proteinfrom serving as a phosphoryl donor in any PTS sugar transport process,thereby blocking further sugar uptake. By mutagenizing HPr such thatthis serine residue cannot be phosphorylated, one may constitutivelyactivate HPr and thereby increase sugar transport into the cell, whichin turn will ensure greater intracellular energy stores andintermediate/precursor molecules for the biosynthesis of one or moredesired fine chemicals.

The isolated nucleic acid sequences of the invention are containedwithin the genome of a Corynebacterium glutamicum strain availablethrough the American Type Culture Collection, given designation ATCC13032. The nucleotide sequence of the isolated C. glutamicum PTS DNAsand the predicted amino acid sequences of the C. glutamicum PTS proteinsare shown in Appendices A and B, respectively. Computational analyseswere performed which classified and/or identified these nucleotidesequences as sequences which encode metabolic pathway proteins.

The present invention also pertains to proteins which have an amino acidsequence which is substantially homologous to an amino acid sequence ofAppendix B. As used herein, a protein which has an amino acid sequencewhich is substantially homologous to a selected amino acid sequence isleast about 50% homologous to the selected amino acid sequence, e.g.,the entire selected amino acid sequence. A protein which has an aminoacid sequence which is substantially homologous to a selected amino acidsequence can also be least about 50-60%, preferably at least about60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%,and most preferably at least about 96%, 97%, 98%, 99% or more homologousto the selected amino acid sequence.

The PTS protein or a biologically active portion or fragment thereof ofthe invention can participate in the transport of high-energycarbon-containing molecules such as glucose into C. glutamicum, or canparticipate in intracellular signal transduction in this microorganism,or may have one or more of the activities set forth in Table 1.

Various aspects of the invention are described in further detail in thefollowing subsections:

A. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode PTS polypeptides or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes or primers for the identification or amplification ofPTS-encoding nucleic acid (e.g., PTS DNA). As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 100 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 20 nucleotidesof sequence downstream from the 3′end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolated PTSnucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived (e.g, a C. glutamicum cell). Moreover, an“isolated” nucleic acid molecule, such as a DNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of Appendix A, or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. For example, a C. glutamicumPTS DNA can be isolated from a C. glutamicum library using all orportion of one of the sequences of Appendix A as a hybridization probeand standard hybridization techniques (e.g., as described in Sambrook,J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleicacid molecule encompassing all or a portion of one of the sequences ofAppendix A can be isolated by the polymerase chain reaction usingoligonucleotide primers designed based upon this sequence (e.g., anucleic acid molecule encompassing all or a portion of one of thesequences of Appendix A can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this same sequence ofAppendix A). For example, mRNA can be isolated from normal endothelialcells (e.g., by the guanidinium-thiocyanate extraction procedure ofChirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA can beprepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for polymerase chain reactionamplification can be designed based upon one of the nucleotide sequencesshown in Appendix A. A nucleic acid of the invention can be amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a PTS nucleotide sequencecan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in Appendix A.The sequences of Appendix A correspond to the Corynebacterium glutamicumPTS DNAs of the invention. This DNA comprises sequences encoding PTSproteins (i.e., the “coding region”, indicated in each sequence inAppendix A), as well as 5′ untranslated sequences and 3′ untranslatedsequences, also indicated in Appendix A. Alternatively, the nucleic acidmolecule can comprise only the coding region of any of the sequences inAppendix A.

For the purposes of this application, it will be understood that each ofthe sequences set forth in Appendix A has an identifying RXA, RXN, RXS,or RXC number having the designation “RXA”, “RXN”, “RXS”, or “RXC”followed by 5 digits (i.e., RXA01503, RXN01299, RXS00315, or RXC00953).Each of these sequences comprises up to three parts: a 5′ upstreamregion, a coding region, and a downstream region. Each of these threeregions is identified by the same RXA, RXN, RXS, or RXC designation toeliminate confusion. The recitation “one of the sequences in AppendixA”, then, refers to any of the sequences in Appendix A, which may bedistinguished by their differing RXA, RXN, RXS, or RXC designations. Thecoding region of each of these sequences is translated into acorresponding amino acid sequence, which is set forth in Appendix B. Thesequences of Appendix B are identified by the same RXA, RXN, RXS, or RXCdesignations as Appendix A, such that they can be readily correlated.For example, the amino acid sequences in Appendix B designated RXA01503,RXN01299, RXS00315, and RXC00953 are translations of the coding regionsof the nucleotide sequence of nucleic acid molecules RXA01503, RXN01299,RXS00315, and RXC00953, respectively, in Appendix A. Each of the RXA,RXN, RXS, and RXC nucleotide and amino acid sequences of the inventionhas also been assigned a SEQ ID NO, as indicated in Table 1. Forexample, as set forth in Table 1, the nucleotide sequence of RXN01299 isSEQ ID NO: 7, and the corresponding amino acid sequence is SEQ ID NO:8.

Several of the genes of the invention are “F-designated genes”. AnF-designated gene includes those genes set forth in Table 1 which havean ‘F’ in front of the RXA, RXN, RXS, or RXC designation. For example,SEQ ID NO:3, designated, as indicated on Table 1, as “F RXA00315”, is anF-designated gene, as are SEQ ID NOs: 9, 11, and 13 (designated on Table1 as “F RXA01299”, “F RXA01883”, and “F RXA01889”, respectively).

In one embodiment, the nucleic acid molecules of the present inventionare not intended to include C. glutamicum those compiled in Table 2. Inthe case of the dapD gene, a sequence for this gene was published inWehrmann, A., et al. (1998) J. Bacteriol. 180(12): 3159-3165. However,the sequence obtained by the inventors of the present application issignificantly longer than the published version. It is believed that thepublished version relied on an incorrect start codon, and thusrepresents only a fragment of the actual coding region.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofone of the nucleotide sequences shown in Appendix A, or a portionthereof. A nucleic acid molecule which is complementary to one of thenucleotide sequences shown in Appendix A is one which is sufficientlycomplementary to one of the nucleotide sequences shown in Appendix Asuch that it can hybridize to one of the nucleotide sequences shown inAppendix A, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleotide sequence which is at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably atleast about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, morepreferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%,92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,98%, 99% or more homologous to a nucleotide sequence shown in AppendixA, or a portion thereof. Ranges and identity values intermediate to theabove-recited ranges, (e.g., 70-90% identical or 80-95% identical) arealso intended to be encompassed by the present invention. For example,ranges of identity values using a combination of any of the above valuesrecited as upper and/or lower limits are intended to be included. In anadditional preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to one of the nucleotidesequences shown in Appendix A, or a portion thereof.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in Appendix A, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of a PTS protein. The nucleotidesequences determined from the cloning of the PTS genes from C.glutamicum allows for the generation of probes and primers designed foruse in identifying and/or cloning PTS homologues in other cell types andorganisms, as well as PTS homologues from other Corynebacteria orrelated species. The probe/primer typically comprises substantiallypurified oligonucleotide. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12, preferably about 25, more preferably about 40, 50or 75 consecutive nucleotides of a sense strand of one of the sequencesset forth in Appendix A, an anti-sense sequence of one of the sequencesset forth in Appendix A, or naturally occurring mutants thereof. Primersbased on a nucleotide sequence of Appendix A can be used in PCRreactions to clone PTS homologues. Probes based on the PTS nucleotidesequences can be used to detect transcripts or genomic sequencesencoding the same or homologous proteins. In preferred embodiments, theprobe further comprises a label group attached thereto, e.g. the labelgroup can be a radioisotope, a fluorescent compound, an enzyme, or anenzyme co-factor. Such probes can be used as a part of a diagnostic testkit for identifying cells which misexpress a PTS protein, such as bymeasuring a level of a PTS-encoding nucleic acid in a sample of cellse.g., detecting PTS mRNA levels or determining whether a genomic PTSgene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of Appendix B suchthat the protein or portion thereof maintains the ability to participatein the transport of high-energy carbon molecules (such as glucose) intoC. glutamicum, and may also participate in one or more intracellularsignal transduction pathways. As used herein, the language “sufficientlyhomologous” refers to proteins or portions thereof which have amino acidsequences which include a minimum number of identical or equivalent(e.g, an amino acid residue which has a similar side chain as an aminoacid residue in one of the sequences of Appendix B) amino acid residuesto an amino acid sequence of Appendix B such that the protein or portionthereof is capable of transporting high-energy carbon-containingmolecules such as glucose into C. glutamicum, and may also participatein intracellular signal transduction in this microorganism. Proteinmembers of such metabolic pathways, as described herein, function totransport high-energy carbon-containing molecules such as glucose intoC. glutamicum, and may also participate in intracellular signaltransduction in this microorganism. Examples of such activities are alsodescribed herein. Thus, “the function of a PTS protein” contributes tothe overall functioning and/or regulation of one or morephosphoenolpyruvate-based sugar transport pathway, and/or contributes,either directly or indirectly, to the yield, production, and/orefficiency of production of one or more fine chemicals. Examples of PTSprotein activities are set forth in Table 1.

In another embodiment, the protein is at least about 50-60%, preferablyat least about 60-70%, and more preferably at least about 70-80%,80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% ormore homologous to an entire amino acid sequence of Appendix B.

Portions of proteins encoded by the PTS nucleic acid molecules of theinvention are preferably biologically active portions of one of the PTSproteins. As used herein, the term “biologically active portion of a PTSprotein” is intended to include a portion, e.g., a domain/motif, of aPTS protein that is capable of transporting high-energycarbon-containing molecules such as glucose into C. glutamicum, or ofparticipating in intracellular signal transduction in thismicroorganism, or has an activity as set forth in Table 1. To determinewhether a PTS protein or a biologically active portion thereof canparticipate in the transportation of high-energy carbon-containingmolecules such as glucose into C. glutamicum, or can participate inintracellular signal transduction in this microorganism, an assay ofenzymatic activity may be performed. Such assay methods are well knownto those of ordinary skill in the art, as detailed in Example 8 of theExemplification.

Additional nucleic acid fragments encoding biologically active portionsof a PTS protein can be prepared by isolating a portion of one of thesequences in Appendix B, expressing the encoded portion of the PTSprotein or peptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the PTS protein orpeptide.

The invention further encompasses nucleic acid molecules that differfrom one of the nucleotide sequences shown in Appendix A (and portionsthereof) due to degeneracy of the genetic code and thus encode the samePTS protein as that encoded by the nucleotide sequences shown inAppendix A. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having anamino acid sequence shown in Appendix B. In a still further embodiment,the nucleic acid molecule of the invention encodes a full length C.glutamicum protein which is substantially homologous to an amino acidsequence of Appendix B (encoded by an open reading frame shown inAppendix A).

It will be understood by one of ordinary skill in the art that in oneembodiment the sequences of the invention are not meant to include thesequences of the prior art, such as those Genbank sequences set forth inTables 2 or 4 which were available prior to the present invention. Inone embodiment, the invention includes nucleotide and amino acidsequences having a percent identity to a nucleotide or amino acidsequence of the invention which is greater than that of a sequence ofthe prior art (e.g., a Genbank sequence (or the protein encoded by sucha sequence) set forth in Tables 2 or 4). For example, the inventionincludes a nucleotide sequence which is greater than and/or at least 44%identical to the nucleotide sequence designated RXA01503 (SEQ ID NO:5),a nucleotide sequence which is greater than and/or at least 41 %identical to the nucleotide sequence designated RXA00951 (SEQ ID NO:15),and a nucleotide sequence which is greater than and/or at least 38%identical to the nucleotide sequence designated RXA01300 (SEQ ID NO:21).One of ordinary skill in the art would be able to calculate the lowerthreshold of percent identity for any given sequence of the invention byexamining the GAP-calculated percent identity scores set forth in Table4 for each of the three top hits for the given sequence, and bysubtracting the highest GAP-calculated percent identity from 100percent. One of ordinary skill in the art will also appreciate thatnucleic acid and amino acid sequences having percent identities greaterthan the lower threshold so calculated (e.g., at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably atleast about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%,and even more preferably at least about 95%, 96%, 97%, 98%, 99% or moreidentical) are also encompassed by the invention.

In addition to the C. glutamicum PTS nucleotide sequences shown inAppendix A, it will be appreciated by those of ordinary skill in the artthat DNA sequence polymorphisms that lead to changes in the amino acidsequences of PTS proteins may exist within a population (e.g., the C.glutamicum population). Such genetic polymorphism in the PTS gene mayexist among individuals within a population due to natural variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a PTS protein,preferably a C. glutamicum PTS protein. Such natural variations cantypically result in 1-5% variance in the nucleotide sequence of the PTSgene. Any and all such nucleotide variations and resulting amino acidpolymorphisms in PTS that are the result of natural variation and thatdo not alter the functional activity of PTS proteins are intended to bewithin the scope of the invention.

Nucleic acid molecules corresponding to natural variants and non-C.glutamicum homologues of the C. glutamicum PTS DNA of the invention canbe isolated based on their homology to the C. glutamicum PTS nucleicacid disclosed herein using the C. glutamicum DNA, or a portion thereof,as a hybridization probe according to standard hybridization techniquesunder stringent hybridization conditions. Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the invention is atleast 15 nucleotides in length and hybridizes under stringent conditionsto the nucleic acid molecule comprising a nucleotide sequence ofAppendix A. In other embodiments, the nucleic acid is at least 30, 50,100, 250 or more nucleotides in length. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 65%, more preferably at least about 70%, andeven more preferably at least about 75% or more homologous to each othertypically remain hybridized to each other. Such stringent conditions areknown to those of ordinary skill in the art and can be found in Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acid moleculeof the invention that hybridizes under stringent conditions to asequence of Appendix A corresponds to a naturally-occurring nucleic acidmolecule. As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural protein). In one embodiment,the nucleic acid encodes a natural C. glutamicum PTS protein.

In addition to naturally-occurring variants of the PTS sequence that mayexist in the population, one of ordinary skill in the art will furtherappreciate that changes can be introduced by mutation into a nucleotidesequence of Appendix A, thereby leading to changes in the amino acidsequence of the encoded PTS protein, without altering the functionalability of the PTS protein. For example, nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues can be made in a sequence of Appendix A. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of one of the PTS proteins (Appendix B) without altering theactivity of said PTS protein, whereas an “essential” amino acid residueis required for PTS protein activity. Other amino acid residues,however, (e.g., those that are not conserved or only semi-conserved inthe domain having PTS activity) may not be essential for activity andthus are likely to be amenable to alteration without altering PTSactivity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding PTS proteins that contain changes in amino acidresidues that are not essential for PTS activity. Such PTS proteinsdiffer in amino acid sequence from a sequence contained in Appendix Byet retain at least one of the PTS activities described herein. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein comprises an amino acidsequence at least about 50% homologous to an amino acid sequence ofAppendix B and is capable of transporting high-energy carbon-containingmolecules such as glucose into C. glutamicum, or of participating inintracellular signal transduction in this microorganism, or has one ormore activities set forth in Table 1. Preferably, the protein encoded bythe nucleic acid molecule is at least about 50-60% homologous to one ofthe sequences in Appendix B, more preferably at least about 60-70%homologous to one of the sequences in Appendix B, even more preferablyat least about 70-80%, 80-90%, 90-95% homologous to one of the sequencesin Appendix B, and most preferably at least about 96%, 97%, 98%, or 99%homologous to one of the sequences in Appendix B.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of Appendix B and a mutant form thereof) or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of one protein or nucleicacid for optimal alignment with the other protein or nucleic acid). Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in onesequence (e.g., one of the sequences of Appendix B) is occupied by thesame amino acid residue or nucleotide as the corresponding position inthe other sequence (e.g., a mutant form of the sequence selected fromAppendix B), then the molecules are homologous at that position (i.e.,as used herein amino acid or nucleic acid “homology” is equivalent toamino acid or nucleic acid “identity”). The percent homology between thetwo sequences is a function of the number of identical positions sharedby the sequences (i.e., % homology=# of identical positions/total # ofpositions×100).

An isolated nucleic acid molecule encoding a PTS protein homologous to aprotein sequence of Appendix B can be created by introducing one or morenucleotide substitutions, additions or deletions into a nucleotidesequence of Appendix A such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into one of the sequences of Appendix A bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a PTS protein is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a PTS coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened fora PTS activity described herein to identify mutants that retain PTSactivity. Following mutagenesis of one of the sequences of Appendix A,the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined using, for example, assays describedherein (see Example 8 of the Exemplification).

In addition to the nucleic acid molecules encoding PTS proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded DNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire PTS coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a PTSprotein. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acid residues(e.g., the entire coding region of SEQ ID NO. 5 (RXA01503) comprisesnucleotides 1 to 249). In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding PTS. The term “noncoding region” refers to5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding PTS disclosed herein (e.g.,the sequences set forth in Appendix A), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of PTS mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of PTS mRNA. For example, the antisense oligonucleotidecan be complementary to the region surrounding the translation startsite of PTS mRNA. An antisense oligonucleotide can be, for example,about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a PTS proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic promoter arepreferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleavePTS mRNA transcripts to thereby inhibit translation of PTS mRNA. Aribozyme having specificity for a PTS-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a PTS DNA disclosedherein (i.e., SEQ ID NO:5 (RXA01503 in Appendix A)). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a PTS-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.5,116,742. Alternatively, PTS mRNA can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, PTS gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a PTSnucleotide sequence (e.g., a PTS promoter and/or enhancers) to formtriple helical structures that prevent transcription of a PTS gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14(12):807-15.

B. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a PTS protein (ora portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells.Preferred regulatory sequences are, for example, promoters such as cos-,tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI^(q)-, T7-, T5-,T3-, gal-, trc-, ara-, SP6-, amy, SPO2, λ-P_(R)- or λ P_(L), which areused preferably in bacteria. Additional regulatory sequences are, forexample, promoters from yeasts and fungi, such as ADC1, MFα, AC, P-60,CYC1, GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S,SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- orphaseolin-promoters. It is also possible to use artificial promoters. Itwill be appreciated by one of ordinary skill in the art that the designof the expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein (e.g., PTSproteins, mutant forms of PTS proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of PTS proteins in prokaryotic or eukaryotic cells. Forexample, PTS genes can be expressed in bacterial cells such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastand other fungal cells (see Romanos, M. A. et al. (1992) “Foreign geneexpression in yeast: a review”, Yeast 8: 423-488; van den Hondel, C. A.M. J. J. et al. (1991) “Heterologous gene expression in filamentousfungi” in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, eds., p.396-428: Academic Press: San Diego; and van den Hondel,C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge University Press:Cambridge), algae and multicellular plant cells (see Schmidt, R. andWillmitzer, L. (1988) High efficiency Agrobacteriumtumefactiens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants” Plant Cell Rep.: 583-586), or mammalian cells.Suitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein. Such fusion vectors typically servethree purposes: 1) to increase expression of recombinant protein; 2) toincrease the solubility of the recombinant protein; and 3) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the PTS protein is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant PTS proteinunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) pLG338, pACYC184,pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200,pUR290, pIN-III1113-B1, λgt11, pBdCl, and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 60-89; and Pouwels et al., eds. (1985) CloningVectors. Elsevier: New York IBSN 0 444 904018). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter. For transformation of other varietiesof bacteria, appropriate vectors may be selected. For example, theplasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful intransforming Streptomyces, while plasmids pUB110, pC194, or pBD214 aresuited for transformation of Bacillus species. Several plasmids of usein the transfer of genetic information into Corynebacterium includepHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds. (1985) CloningVectors. Elsevier: New York IBSN 0 444 904018).

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in the bacterium chosen for expression, such asC. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

In another embodiment, the PTS protein expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234),2μ, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982)Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), andpYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methodsfor the construction of vectors appropriate for use in other fungi, suchas the filamentous fungi, include those detailed in: van den Hondel, C.A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press:Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier:New York (IBSN 0 444 904018).

Alternatively, the PTS proteins of the invention can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In another embodiment, the PTS proteins of the invention may beexpressed in unicellular plant cells (such as algae) or in plant cellsfrom higher plants (e.g., the spermatophytes, such as crop plants).Examples of plant expression vectors include those detailed in: Becker,D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plant binaryvectors with selectable markers located proximal to the left border”,Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation”, Nucl. Acid. Res. 12:8711-8721, and include pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51(Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0444 904018).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PTS mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aPTS protein can be expressed in bacterial cells such as C. glutamicum,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to one ofordinary skill in the art. Microorganisms related to Corynebacteriumglutamicum which may be conveniently used as host cells for the nucleicacid and protein molecules of the invention are set forth in Table 3.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., linear DNA or RNA (e.g., a linearized vector or a geneconstruct alone without a vector) or nucleic acid in the form of avector (e.g., a plasmid, phage, phasmid, phagemid, transposon or otherDNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (Molecular Cloning: A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a PTS protein or can be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is preparedwhich contains at least a portion of a PTS gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PTS gene. Preferably, this PTS gene is aCorynebacterium glutamicum PTS gene, but it can be a homologue from arelated bacterium or even from a mammalian, yeast, or insect source. Ina preferred embodiment, the vector is designed such that, uponhomologous recombination, the endogenous PTS gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a “knock out” vector). Alternatively, the vector can be designedsuch that, upon homologous recombination, the endogenous PTS gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous PTS protein). In the homologousrecombination vector, the altered portion of the PTS gene is flanked atits 5′ and 3′ ends by additional nucleic acid of the PTS gene to allowfor homologous recombination to occur between the exogenous PTS genecarried by the vector and an endogenous PTS gene in a microorganism. Theadditional flanking PTS nucleic acid is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see e.g., Thomas, K. R., and Capecchi, M. R.(1987) Cell 51: 503 for a description of homologous recombinationvectors). The vector is introduced into a microorganism (e.g., byelectroporation) and cells in which the introduced PTS gene hashomologously recombined with the endogenous PTS gene are selected, usingart-known techniques.

In another embodiment, recombinant microorganisms can be produced whichcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of a PTS gene on a vectorplacing it under control of the lac operon permits expression of the PTSgene only in the presence of IPTG. Such regulatory systems are wellknown in the art.

In another embodiment, an endogenous PTS gene in a host cell isdisrupted (e.g., by homologous recombination or other genetic meansknown in the art) such that expression of its protein product does notoccur. In another embodiment, an endogenous or introduced PTS gene in ahost cell has been altered by one or more point mutations, deletions, orinversions, but still encodes a functional PTS protein. In still anotherembodiment, one or more of the regulatory regions (e.g., a promoter,repressor, or inducer) of a PTS gene in a microorganism has been altered(e.g., by deletion, truncation, inversion, or point mutation) such thatthe expression of the PTS gene is modulated. One of ordinary skill inthe art will appreciate that host cells containing more than one of thedescribed PTS gene and protein modifications may be readily producedusing the methods of the invention, and are meant to be included in thepresent invention.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a PTS protein.Accordingly, the invention further provides methods for producing PTSproteins using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding a PTS protein has beenintroduced, or into which genome has been introduced a gene encoding awild-type or altered PTS protein) in a suitable medium until PTS proteinis produced. In another embodiment, the method further comprisesisolating PTS proteins from the medium or the host cell.

C. Isolated PTS Proteins

Another aspect of the invention pertains to isolated PTS proteins, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is substantially free ofcellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof PTS protein in which the protein is separated from cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of PTS protein having less thanabout 30% (by dry weight) of non-PTS protein (also referred to herein asa “contaminating protein”), more preferably less than about 20% ofnon-PTS protein, still more preferably less than about 10% of non-PTSprotein, and most preferably less than about 5% non-PTS protein. Whenthe PTS protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation. The language “substantially free ofchemical precursors or other chemicals” includes preparations of PTSprotein in which the protein is separated from chemical precursors orother chemicals which are involved in the synthesis of the protein. Inone embodiment, the language “substantially free of chemical precursorsor other chemicals” includes preparations of PTS protein having lessthan about 30% (by dry weight) of chemical precursors or non-PTSchemicals, more preferably less than about 20% chemical precursors ornon-PTS chemicals, still more preferably less than about 10% chemicalprecursors or non-PTS chemicals, and most preferably less than about 5%chemical precursors or non-PTS chemicals. In preferred embodiments,isolated proteins or biologically active portions thereof lackcontaminating proteins from the same organism from which the PTS proteinis derived. Typically, such proteins are produced by recombinantexpression of, for example, a C. glutamicum PTS protein in amicroorganism such as C. glutamicum.

An isolated PTS protein or a portion thereof of the invention canparticipate in the transport of high-energy carbon-containing moleculessuch as glucose into C. glutamicum, and may also participate inintracellular signal transduction in this microorganism, or has one ormore of the activities set forth in Table 1. In preferred embodiments,the protein or portion thereof comprises an amino acid sequence which issufficiently homologous to an amino acid sequence of Appendix B suchthat the protein or portion thereof maintains the ability to transporthigh-energy carbon-containing molecules such as glucose into C.glutamicum, or to participate in intracellular signal transduction inthis microorganism. The portion of the protein is preferably abiologically active portion as described herein. In another preferredembodiment, a PTS protein of the invention has an amino acid sequenceshown in Appendix B. In yet another preferred embodiment, the PTSprotein has an amino acid sequence which is encoded by a nucleotidesequence which hybridizes, e.g., hybridizes under stringent conditions,to a nucleotide sequence of Appendix A. In still another preferredembodiment, the PTS protein has an amino acid sequence which is encodedby a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at leastabout 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or 91%, 92%, 93%, 94%, andeven more preferably at least about 95%, 96%, 97%, 98%, 99% or morehomologous to one of the nucleic acid sequences of Appendix A, or aportion thereof. Ranges and identity values intermediate to theabove-recited values, (e.g., 70-90% identical or 80-95% identical) arealso intended to be encompassed by the present invention. For example,ranges of identity values using a combination of any of the above valuesrecited as upper and/or lower limits are intended to be included. Thepreferred PTS proteins of the present invention also preferably possessat least one of the PTS activities described herein. For example, apreferred PTS protein of the present invention includes an amino acidsequence encoded by a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to a nucleotide sequence ofAppendix A, and which can participate in the transport of high-energycarbon-containing molecules such as glucose into C. glutamicum, and mayalso participate in intracellular signal transduction in thismicroorganism, or which has one or more of the activities set forth inTable 1.

In other embodiments, the PTS protein is substantially homologous to anamino acid sequence of Appendix B and retains the functional activity ofthe protein of one of the sequences of Appendix B yet differs in aminoacid sequence due to natural variation or mutagenesis, as described indetail in subsection I above. Accordingly, in another embodiment, thePTS protein is a protein which comprises an amino acid sequence which isat least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%,preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%,96%, 97%, 98%, 99% or more homologous to an entire amino acid sequenceof Appendix B and which has at least one of the PTS activities describedherein. Ranges and identity values intermediate to the above-recitedvalues, (e.g., 70-90% identical or 80-95% identical) are also intendedto be encompassed by the present invention. For example, ranges ofidentity values using a combination of any of the above values recitedas upper and/or lower limits are intended to be included. In anotherembodiment, the invention pertains to a full length C. glutamicumprotein which is substantially homologous to an entire amino acidsequence of Appendix B.

Biologically active portions of a PTS protein include peptidescomprising amino acid sequences derived from the amino acid sequence ofa PTS protein, e.g., the an amino acid sequence shown in Appendix B orthe amino acid sequence of a protein homologous to a PTS protein, whichinclude fewer amino acids than a full length PTS protein or the fulllength protein which is homologous to a PTS protein, and exhibit atleast one activity of a PTS protein. Typically, biologically activeportions (peptides, e.g., peptides which are, for example, 5, 10, 15,20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length)comprise a domain or motif with at least one activity of a PTS protein.Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the activities described herein.Preferably, the biologically active portions of a PTS protein includeone or more selected domains/motifs or portions thereof havingbiological activity.

PTS proteins are preferably produced by recombinant DNA techniques. Forexample, a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the PTS protein isexpressed in the host cell. The PTS protein can then be isolated fromthe cells by an appropriate purification scheme using standard proteinpurification techniques. Alternative to recombinant expression, a PTSprotein, polypeptide, or peptide can be synthesized chemically usingstandard peptide synthesis techniques. Moreover, native PTS protein canbe isolated from cells (e.g., endothelial cells), for example using ananti-PTS antibody, which can be produced by standard techniquesutilizing a PTS protein or fragment thereof of this invention.

The invention also provides PTS chimeric or fusion proteins. As usedherein, a PTS “chimeric protein” or “fusion protein” comprises a PTSpolypeptide operatively linked to a non-PTS polypeptide. An “PTSpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to PTS, whereas a “non-PTS polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the PTS protein, e.g., aprotein which is different from the PTS protein and which is derivedfrom the same or a different organism. Within the fusion protein, theterm “operatively linked” is intended to indicate that the PTSpolypeptide and the non-PTS polypeptide are fused in-frame to eachother. The non-PTS polypeptide can be fused to the N-terminus orC-terminus of the PTS polypeptide. For example, in one embodiment thefusion protein is a GST-PTS fusion protein in which the PTS sequencesare fused to the C-terminus of the GST sequences. Such fusion proteinscan facilitate the purification of recombinant PTS proteins. In anotherembodiment, the fusion protein is a PTS protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of a PTSprotein can be increased through use of a heterologous signal sequence.

Preferably, a PTS chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A PTS-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the PTS protein.

Homologues of the PTS protein can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the PTS protein. As usedherein, the term “homologue” refers to a variant form of the PTS proteinwhich acts as an agonist or antagonist of the activity of the PTSprotein. An agonist of the PTS protein can retain substantially thesame, or a subset, of the biological activities of the PTS protein. Anantagonist of the PTS protein can inhibit one or more of the activitiesof the naturally occurring form of the PTS protein, by, for example,competitively binding to a downstream or upstream member of the PTSsystem which includes the PTS protein. Thus, the C. glutamicum PTSprotein and homologues thereof of the present invention may modulate theactivity of one or more sugar transport pathways or intracellular signaltransduction pathways in which PTS proteins play a role in thismicroorganism.

In an alternative embodiment, homologues of the PTS protein can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the PTS protein for PTS protein agonist orantagonist activity. In one embodiment, a variegated library of PTSvariants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof PTS variants can be produced by, for example, enzymatically ligatinga mixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential PTS sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of PTS sequences therein.There are a variety of methods which can be used to produce libraries ofpotential PTS homologues from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential PTS sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477.

In addition, libraries of fragments of the PTS protein coding can beused to generate a variegated population of PTS fragments for screeningand subsequent selection of homologues of a PTS protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a PTS coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PTS protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of PTS homologues. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify PTS homologues (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In another embodiment, cell based assays can be exploited to analyze avariegated PTS library, using methods well known in the art.

D. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification of C. glutamicumand related organisms; mapping of genomes of organisms related to C.glutamicum; identification and localization of C. glutamicum sequencesof interest; evolutionary studies; determination of PTS protein regionsrequired for function; modulation of a PTS protein activity; modulationof the activity of a PTS pathway; and modulation of cellular productionof a desired compound, such as a fine chemical.

The PTS nucleic acid molecules of the invention have a variety of uses.First, they may be used to identify an organism as being Corynebacteriumglutamicum or a close relative thereof. Also, they may be used toidentify the presence of C. glutamicum or a relative thereof in a mixedpopulation of microorganisms. The invention provides the nucleic acidsequences of a number of C. glutamicum genes; by probing the extractedgenomic DNA of a culture of a unique or mixed population ofmicroorganisms under stringent conditions with a probe spanning a regionof a C. glutamicum gene which is unique to this organism, one canascertain whether this organism is present.

Although Corynebacterium glutamicum itself is nonpathogenic, it isrelated to pathogenic species, such as Corynebacterium diphtheriae.Corynebacterium diphtheriae is the causative agent of diphtheria, arapidly developing, acute, febrile infection which involves both localand systemic pathology. In this disease, a local lesion develops in theupper respiratory tract and involves necrotic injury to epithelialcells; the bacilli secrete toxin which is disseminated through thislesion to distal susceptible tissues of the body. Degenerative changesbrought about by the inhibition of protein synthesis in these tissues,which include heart, muscle, peripheral nerves, adrenals, kidneys, liverand spleen, result in the systemic pathology of the disease. Diphtheriacontinues to have high incidence in many parts of the world, includingAfrica, Asia, Eastern Europe and the independent states of the formerSoviet Union. An ongoing epidemic of diphtheria in the latter tworegions has resulted in at least 5,000 deaths since 1990.

In one embodiment, the invention provides a method of identifying thepresence or activity of Cornyebacterium diphtheriae in a subject. Thismethod includes detection of one or more of the nucleic acid or aminoacid sequences of the invention (e.g., the sequences set forth inAppendix A or Appendix B) in a subject, thereby detecting the presenceor activity of Corynebacterium diphtheriae in the subject. C. glutamicumand C. diphtheriae are related bacteria, and many of the nucleic acidand protein molecules in C. glutamicum are homologous to C. diphtheriaenucleic acid and protein molecules, and can therefore be used to detectC. diphtheriae in a subject.

The nucleic acid and protein molecules of the invention may also serveas markers for specific regions of the genome. This has utility not onlyin the mapping of the genome, but also for functional studies of C.glutamicum proteins. For example, to identify the region of the genometo which a particular C. glutamicum DNA-binding protein binds, the C.glutamicum genome could be digested, and the fragments incubated withthe DNA-binding protein. Those which bind the protein may beadditionally probed with the nucleic acid molecules of the invention,preferably with readily detectable labels; binding of such a nucleicacid molecule to the genome fragment enables the localization of thefragment to the genome map of C. glutamicum, and, when performedmultiple times with different enzymes, facilitates a rapid determinationof the nucleic acid sequence to which the protein binds. Further, thenucleic acid molecules of the invention may be sufficiently homologousto the sequences of related species such that these nucleic acidmolecules may serve as markers for the construction of a genomic map inrelated bacteria, such as Brevibacterium lactofermentum.

The PTS nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The sugar uptake system inwhich the molecules of the invention participate are utilized by a widevariety of bacteria; by comparing the sequences of the nucleic acidmolecules of the present invention to those encoding similar enzymesfrom other organisms, the evolutionary relatedness of the organisms canbe assessed. Similarly, such a comparison permits an assessment of whichregions of the sequence are conserved and which are not, which may aidin determining those regions of the protein which are essential for thefunctioning of the enzyme. This type of determination is of value forprotein engineering studies and may give an indication of what theprotein can tolerate in terms of mutagenesis without losing function.

Manipulation of the PTS nucleic acid molecules of the invention mayresult in the production of PTS proteins having functional differencesfrom the wild-type PTS proteins. These proteins may be improved inefficiency or activity, may be present in greater numbers in the cellthan is usual, or may be decreased in efficiency or activity.

The invention provides methods for screening molecules which modulatethe activity of a PTS protein, either by interacting with the proteinitself or a substrate or binding partner of the PTS protein, or bymodulating the transcription or translation of a PTS nucleic acidmolecule of the invention. In such methods, a microorganism expressingone or more PTS proteins of the invention is contacted with one or moretest compounds, and the effect of each test compound on the activity orlevel of expression of the PTS protein is assessed.

The PTS molecules of the invention may be modified such that the yield,production, and/or efficiency of production of one or more finechemicals is improved. For example, by modifying a PTS protein involvedin the uptake of glucose such that it is optimized in activity, thequantity of glucose uptake or the rate at which glucose is translocatedinto the cell may be increased. The breakdown of glucose and othersugars within the cell provides energy that may be used to driveenergetically unfavorable biochemical reactions, such as those involvedin the biosynthesis of fine chemicals. This breakdown also providesintermediate and precursor molecules necessary for the biosynthesis ofcertain fine chemicals, such as amino acids, vitamins and cofactors. Byincreasing the amount of intracellular high-energy carbon moleculesthrough modification of the PTS molecules of the invention, one maytherefore increase both the energy available to perform metabolicpathways necessary for the production of one or more fine chemicals, andalso the intracellular pools of metabolites necessary for suchproduction. Conversely, by decreasing the importation of a sugar whosebreakdown products include a compound which is used solely in metabolicpathways which compete with pathways utilized for the production of adesired fine chemical for enzymes, cofactors, or intermediates, one maydownregulate this pathway and thus perhaps increase production throughthe desired biosynthetic pathway.

Further, the PTS molecules of the invention may be involved in one ormore intracellular signal transduction pathways which may affect theyields and/or rate of production of one or more fine chemical from C.glutamicum. For example, proteins necessary for the import of one ormore sugars from the extracellular medium (e.g., HPr, Enzyme I, or amember of an Enzyme II complex) are frequently posttranslationallymodified upon the presence of a sufficient quantity of the sugar in thecell, such that they are no longer able to import that sugar. An exampleof this occurs in E. coli, where high intracellular levels of fructose1,6-bisphosphate result in the phosphorylation of HPr at serine-46, uponwhich this molecule is no longer able to participate in the transport ofany sugar. While this intracellular level of sugar at which thetransport system is shut off may be sufficient to sustain the normalfunctioning of the cell, it may be limiting for the overproduction ofthe desired fine chemical. Thus, it may be desirable to modify the PTSproteins of the invention such that they are no longer responsive tosuch negative regulation, thereby permitting greater intracellularconcentrations of one or more sugars to be achieved, and, by extension,more efficient production or greater yields of one or more finechemicals from organisms containing such mutant PTS proteins.

This aforementioned list of mutagenesis strategies for PTS proteins toresult in increased yields of a desired compound is not meant to belimiting; variations on these mutagenesis strategies will be readilyapparent to one of ordinary skill in the art. By these mechanisms, thenucleic acid and protein molecules of the invention may be utilized togenerate C. glutamicum or related strains of bacteria expressing mutatedPTS nucleic acid and protein molecules such that the yield, production,and/or efficiency of production of a desired compound is improved. Thisdesired compound may be any natural product of C. glutamicum, whichincludes the final products of biosynthesis pathways and intermediatesof naturally-occurring metabolic pathways, as well as molecules which donot naturally occur in the metabolism of C. glutamicum, but which areproduced by a C. glutamicum strain of the invention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patent applications, patents, published patent applications, Tables,Appendices, and the sequence listing cited throughout this applicationare hereby incorporated by reference.

Exemplification

EXAMPLE 1 Preparation of Total Genomic DNA of Corynebacterium glutamicumATCC 13032

A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnightat 30° C. with vigorous shaking in BHI medium (Difco). The cells wereharvested by centrifugation, the supernatant was discarded and the cellswere resuspended in 5 ml buffer-I (5% of the original volume of theculture—all indicated volumes have been calculated for 100 ml of culturevolume). Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/lMgSO₄×7H₂O, 10 ml/l KH₂PO₄ solution (100 g/l, adjusted to pH 6.7 withKOH), 50 ml/l M12 concentrate (10 g/l (NH₄)₂SO₄, 1 g/l NaCl, 2 g/lMgSO₄×7H₂O, 0.2 g/l CaCl₂, 0.5 g/l yeast extract (Difco), 10 ml/ltrace-elements-mix (200 mg/l FeSO₄×H₂O, 10 mg/l ZnSO₄×7 H₂O, 3 mg/lMnCl₂×4 H₂O, 30 mg/l H₃BO₃ 20 mg/l CoCl₂×6 H₂O, 1 mg/l NiCl₂×6 H₂O, 3mg/l Na₂MoO₄×2 H₂O, 500 mg/l complexing agent (EDTA or critic acid), 100ml/l vitamins-mix (0.2 mg/l biotin, 0.2 mg/l folic acid, 20 mg/l p-aminobenzoic acid, 20 mg/l riboflavin, 40 mg/l ca-panthothenate, 140 mg/lnicotinic acid, 40 mg/l pyridoxole hydrochloride, 200 mg/lmyo-inositol). Lysozyme was added to the suspension to a finalconcentration of 2.5 mg/ml. After an approximately 4 h incubation at 37°C., the cell wall was degraded and the resulting protoplasts areharvested by centrifugation. The pellet was washed once with 5 mlbuffer-I and once with 5 ml TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8).The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution(10%) and 0.5 ml NaCl solution (5 M) are added. After adding ofproteinase K to a final concentration of 200 μg/ml, the suspension isincubated for ca. 18 h at 37° C. The DNA was purified by extraction withphenol, phenol-chloroform-isoamylalcohol and chloroform-isoamylalcoholusing standard procedures. Then, the DNA was precipitated by adding 1/50volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30min incubation at −20° C. and a 30 min centrifugation at 12,000 rpm in ahigh speed centrifuge using a SS34 rotor (Sorvall). The DNA wasdissolved in 1 ml TE-buffer containing 20 μg/ml RNaseA and dialysed at4° C. against 1000 ml TE-buffer for at least 3 hours. During this time,the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysedDNA solution, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol are added. Aftera 30 min incubation at −20° C., the DNA was collected by centrifugation(13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pelletwas dissolved in TE-buffer. DNA prepared by this procedure could be usedfor all purposes, including southern blotting or construction of genomiclibraries.

EXAMPLE 2 Construction of Genomic Libraries in Escherichia coli ofCorynebacterium glutamicum ATCC13032.

Using DNA prepared as described in Example 1, cosmid and plasmidlibraries were constructed according to known and well establishedmethods (see e.g., Sambrook, J. et al. (1989) “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons.)

Any plasmid or cosmid could be used. Of particular use were the plasmidspBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci. USA,75:3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK− and others;Stratagene, LaJolla, USA), or cosmids as SuperCos1 (Stratagene, LaJolla,USA) or Lorist6 (Gibson, T. J., Rosenthal A. and Waterson, R. H. (1987)Gene 53:283-286. Gene libraries specifically for use in C. glutamicummay be constructed using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey(1994) J. Microbiol. Biotechnol. 4: 256-263).

EXAMPLE 3 DNA Sequencing and Computational Functional Analysis

Genomic libraries as described in Example 2 were used for DNA sequencingaccording to standard methods, in particular by the chain terminationmethod using ABI377 sequencing machines (see e.g., Fleischman, R. D. etal. (1995) “Whole-genome Random Sequencing and Assembly of HaemophilusInfluenzae Rd., Science, 269:496-512). Sequencing primers with thefollowing nucleotide sequences were used: 5′-GGAAACAGTATGACCATG-3′ or5′-GTAAAACGACGGCCAGT-3′.

EXAMPLE 4 In vivo Mutagenesis

In vivo mutagenesis of Corynebacterium glutamicum can be performed bypassage of plasmid (or other vector) DNA through E. coli or othermicroorganisms (e.g. Bacillus spp. or yeasts such as Saccharomycescerevisiae) which are impaired in their capabilities to maintain theintegrity of their genetic information. Typical mutator strains havemutations in the genes for the DNA repair system (e.g., mutHLS, mutD,mutT, etc.; for reference, see Rupp, W. D. (1996) DNA repair mechanisms,in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.)Such strains are well known to one of ordinary skill in the art. The useof such strains is illustrated, for example, in Greener, A. andCallahan, M. (1994) Strategies 7: 32-34.

EXAMPLE 5 DNA Transfer Between Escherichia coli and Corynebacteriumglutamicum

Several Corynebacterium and Brevibacterium species contain endogenousplasmids (as e.g., pHM1519 or pBL1) which replicate autonomously (forreview see, e.g., Martin, J. F. et al. (1987) Biotechnology, 5:137-146).Shuttle vectors for Escherichia coli and Corynebacterium glutamicum canbe readily constructed by using standard vectors for E. coli (Sambrook,J. et al. (1989), “Molecular Cloning: A Laboratory Manual”, Cold SpringHarbor Laboratory Press or Ausubel, F. M. et al. (1994) “CurrentProtocols in Molecular Biology”, John Wiley & Sons) to which a origin orreplication for and a suitable marker from Corynebacterium glutamicum isadded. Such origins of replication are preferably taken from endogenousplasmids isolated from Corynebacterium and Brevibacterium species. Ofparticular use as transformation markers for these species are genes forkanamycin resistance (such as those derived from the Tn5 or Tn903transposons) or chloramphenicol (Winnacker, E. L. (1987) “From Genes toClones—Introduction to Gene Technology, VCH, Weinheim). There arenumerous examples in the literature of the construction of a widevariety of shuttle vectors which replicate in both E. coli and C.glutamicum, and which can be used for several purposes, including geneover-expression (for reference, see e.g., Yoshihama, M. et al. (1985) J.Bacteriol. 162:591-597, Martin J. F. et al. (1987) Biotechnology,5:137-146 and Eikmanns, B. J. et al. (1991) Gene, 102:93-98).

Using standard methods, it is possible to clone a gene of interest intoone of the shuttle vectors described above and to introduce such ahybrid vectors into strains of Corynebacterium glutamicum.Transformation of C. glutamicum can be achieved by protoplasttransformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311),electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters,53:399-303) and in cases where special vectors are used, also byconjugation (as described e.g. in Schäfer, A et al. (1990) J. Bacteriol.172:1663-1666). It is also possible to transfer the shuttle vectors forC. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum(using standard methods well-known in the art) and transforming it intoE. coli. This transformation step can be performed using standardmethods, but it is advantageous to use an Mcr-deficient E. coli strain,such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).

Genes may be overexpressed in C. glutamicum strains using plasmids whichcomprise pCG1 (U.S. Pat. No. 4,617,267) or fragments thereof, andoptionally the gene for kanamycin resistance from TN903 (Grindley, N. D.and Joyce, C. M. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180).In addition, genes may be overexpressed in C. glutamicum strains usingplasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J. Microbiol.Biotechnol. 4: 256-263).

Aside from the use of replicative plasmids, gene overexpression can alsobe achieved by integration into the genome. Genomic integration in C.glutamicum or other Corynebacterium or Brevibacterium species may beaccomplished by well-known methods, such as homologous recombinationwith genomic region(s), restriction endonuclease mediated integration(REMI) (see, e.g., DE Patent 19823834), or through the use oftransposons. It is also possible to modulate the activity of a gene ofinterest by modifying the regulatory regions (e.g., a promoter, arepressor, and/or an enhancer) by sequence modification, insertion, ordeletion using site-directed methods (such as homologous recombination)or methods based on random events (such as transposon mutagenesis orREMI). Nucleic acid sequences which function as transcriptionalterminators may also be inserted 3′ to the coding region of one or moregenes of the invention; such terminators are well-known in the art andare described, for example, in Winnacker, E. L. (1987) From Genes toClones—Introduction to Gene Technology. VCH: Weinheim.

EXAMPLE 6 Assessment of the Expression of the Mutant Protein

Observations of the activity of a mutated protein in a transformed hostcell rely on the fact that the mutant protein is expressed in a similarfashion and in a similar quantity to that of the wild-type protein. Auseful method to ascertain the level of transcription of the mutant gene(an indicator of the amount of mRNA available for translation to thegene product) is to perform a Northern blot (for reference see, forexample, Ausubel et al. (1988) Current Protocols in Molecular Biology,Wiley: New York), in which a primer designed to bind to the gene ofinterest is labeled with a detectable tag (usually radioactive orchemiluminescent), such that when the total RNA of a culture of theorganism is extracted, run on gel, transferred to a stable matrix andincubated with this probe, the binding and quantity of binding of theprobe indicates the presence and also the quantity of mRNA for thisgene. This information is evidence of the degree of transcription of themutant gene. Total cellular RNA can be prepared from Corynebacteriumglutamicum by several methods, all well-known in the art, such as thatdescribed in Bormann, E. R. et al. (1992) Mol. Microbiol. 6: 317-326.

To assess the presence or relative quantity of protein translated fromthis mRNA, standard techniques, such as a Western blot, may be employed(see, for example, Ausubel et al. (1988) Current Protocols in MolecularBiology, Wiley: New York). In this process, total cellular proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose, and incubated with a probe, such as an antibody,which specifically binds to the desired protein. This probe is generallytagged with a chemiluminescent or colorimetric label which may bereadily detected. The presence and quantity of label observed indicatesthe presence and quantity of the desired mutant protein present in thecell.

EXAMPLE 7 Growth of Genetically Modified Corynebacteriumglutamicum—Media and Culture Conditions

Genetically modified Corynebacteria are cultured in synthetic or naturalgrowth media. A number of different growth media for Corynebacteria areboth well-known and readily available (Lieb et al. (1989) Appl.Microbiol. Biotechnol., 32:205-210; von der Osten et al. (1998)Biotechnology Letters, 11:11-16; Pat. DE 4,120,867; Liebl (1992) “TheGenus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. etal., eds. Springer-Verlag). These media consist of one or more carbonsources, nitrogen sources, inorganic salts, vitamins and trace elements.Preferred carbon sources are sugars, such as mono-, di-, orpolysaccharides. For example, glucose, fructose, mannose, galactose,ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starchor cellulose serve as very good carbon sources. It is also possible tosupply sugar to the media via complex compounds such as molasses orother by-products from sugar refinement. It can also be advantageous tosupply mixtures of different carbon sources. Other possible carbonsources are alcohols and organic acids, such as methanol, ethanol,acetic acid or lactic acid. Nitrogen sources are usually organic orinorganic nitrogen compounds, or materials which contain thesecompounds. Exemplary nitrogen sources include ammonia gas or ammoniasalts, such as NH₄Cl or (NH₄)₂SO₄, NH₄OH, nitrates, urea, amino acids orcomplex nitrogen sources like corn steep liquor, soy bean flour, soybean protein, yeast extract, meat extract and others.

Inorganic salt compounds which may be included in the media include thechloride-, phosphorous- or sulfate-salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Chelating compounds can be added to the medium to keep the metal ions insolution. Particularly useful chelating compounds includedihydroxyphenols, like catechol or protocatechuate, or organic acids,such as citric acid. It is typical for the media to also contain othergrowth factors, such as vitamins or growth promoters, examples of whichinclude biotin, riboflavin, thiamin, folic acid, nicotinic acid,pantothenate and pyridoxin. Growth factors and salts frequentlyoriginate from complex media components such as yeast extract, molasses,corn steep liquor and others. The exact composition of the mediacompounds depends strongly on the immediate experiment and isindividually decided for each specific case. Information about mediaoptimization is available in the textbook “Applied Microbiol.Physiology, A Practical Approach (eds. P. M. Rhodes, P. F. Stanbury, IRLPress (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible toselect growth media from commercial suppliers, like standard 1 (Merck)or BHI (grain heart infusion, DIFCO) or others.

All medium components are sterilized, either by heat (20 minutes at 1.5bar and 121° C.) or by sterile filtration. The components can either besterilized together or, if necessary, separately. All media componentscan be present at the beginning of growth, or they can optionally beadded continuously or batchwise.

Culture conditions are defined separately for each experiment. Thetemperature should be in a range between 15° C. and 45° C. Thetemperature can be kept constant or can be altered during theexperiment. The pH of the medium should be in the range of 5 to 8.5,preferably around 7.0, and can be maintained by the addition of buffersto the media. An exemplary buffer for this purpose is a potassiumphosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and otherscan alternatively or simultaneously be used. It is also possible tomaintain a constant culture pH through the addition of NaOH or NH₄OHduring growth. If complex medium components such as yeast extract areutilized, the necessity for additional buffers may be reduced, due tothe fact that many complex compounds have high buffer capacities. If afermentor is utilized for culturing the micro-organisms, the pH can alsobe controlled using gaseous ammonia.

The incubation time is usually in a range from several hours to severaldays. This time is selected in order to permit the maximal amount ofproduct to accumulate in the broth. The disclosed growth experiments canbe carried out in a variety of vessels, such as microtiter plates, glasstubes, glass flasks or glass or metal fermentors of different sizes. Forscreening a large number of clones, the microorganisms should becultured in microtiter plates, glass tubes or shake flasks, either withor without baffles. Preferably 100 ml shake flasks are used, filled with10% (by volume) of the required growth medium. The flasks should beshaken on a rotary shaker (amplitude 25 mm) using a speed-range of100-300 rpm. Evaporation losses can be diminished by the maintenance ofa humid atmosphere; alternatively, a mathematical correction forevaporation losses should be performed.

If genetically modified clones are tested, an unmodified control cloneor a control clone containing the basic plasmid without any insertshould also be tested. The medium is inoculated to an OD₆₀₀ of 0.5-1.5using cells grown on agar plates, such as CM plates (10 g/l glucose, 2.5g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/lmeat extract, 22 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeastextract, 5 g/l meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that hadbeen incubated at 30° C. Inoculation of the media is accomplished byeither introduction of a saline suspension of C. glutamicum cells fromCM plates or addition of a liquid preculture of this bacterium.

EXAMPLE 8 In vitro Analysis of the Function of Mutant Proteins

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one ofordinary skill in the art. Overviews about enzymes in general, as wellas specific details concerning structure, kinetics, principles, methods,applications and examples for the determination of many enzymeactivities may be found, for example, in the following references:Dixon, M., and Webb, E. C., (1979) Enzymes. Longmans: London; Fersht,(1985) Enzyme Structure and Mechanism. Freeman: New York; Walsh, (1979)Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N. C.,Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press:Oxford; Boyer, P. D., ed. (1983) The Enzymes, 3^(rd) ed. Academic Press:New York; Bisswanger, H., (1994) Enzymkinetik, 2^(nd) ed. VCH: Weinheim(ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβ1, M., eds.(1983-1986) Methods of Enzymatic Analysis, 3^(rd) ed., vol. I-XII,Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of IndustrialChemistry (1987) vol. A9, “Enzymes”. VCH: Weinheim, p. 352-363.

The activity of proteins which bind to DNA can be measured by severalwell-established methods, such as DNA band-shift assays (also called gelretardation assays). The effect of such proteins on the expression ofother molecules can be measured using reporter gene assays (such as thatdescribed in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 andreferences cited therein). Reporter gene test systems are well known andestablished for applications in both pro- and eukaryotic cells, usingenzymes such as beta-galactosidase, green fluorescent protein, andseveral others.

The determination of activity of membrane-transport proteins can beperformed according to techniques such as those described in Gennis, R.B. (1989) “Pores, Channels and Transporters”, in Biomembranes, MolecularStructure and Function, Springer: Heidelberg, p. 85-137; 199-234; and270-322.

EXAMPLE 9 Analysis of Impact of Mutant Protein on the Production of theDesired Product

The effect of the genetic modification in C. glutamicum on production ofa desired compound (such as an amino acid) can be assessed by growingthe modified microorganism under suitable conditions (such as thosedescribed above) and analyzing the medium and/or the cellular componentfor increased production of the desired product (i.e., an amino acid).Such analysis techniques are well known to one of ordinary skill in theart, and include spectroscopy, thin layer chromatography, stainingmethods of various kinds, enzymatic and microbiological methods, andanalytical chromatography such as high performance liquid chromatography(see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol.A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al.,(1987) “Applications of HPLC in Biochemistry” in: Laboratory Techniquesin Biochemistry and Molecular Biology, vol. 17; Rehm et al. (1993)Biotechnology, vol. 3, Chapter III: “Product recovery and purification”,page 469-714, VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations:downstream processing for biotechnology, John Wiley and Sons; Kennedy,J. F. and Cabral, J. M. S. (1992) Recovery processes for biologicalmaterials, John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988)Biochemical separations, in: Ulmann's Encyclopedia of IndustrialChemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications.)

In addition to the measurement of the final product of fermentation, itis also possible to analyze other components of the metabolic pathwaysutilized for the production of the desired compound, such asintermediates and side-products, to determine the overall productivityof the organism, yield, and/or efficiency of production of the compound.Analysis methods include measurements of nutrient levels in the medium(e.g., sugars, hydrocarbons, nitrogen sources, phosphate, and otherions), measurements of biomass composition and growth, analysis of theproduction of common metabolites of biosynthetic pathways, andmeasurement of gasses produced during fermentation. Standard methods forthese measurements are outlined in Applied Microbial Physiology, APractical Approach, P. M. Rhodes and P. F. Stanbury, eds., IRL Press, p.103-129; 131-163; and 165-192 (ISBN: 0199635773) and references citedtherein.

EXAMPLE 10 Purification of the Desired Product from C. glutamicumCulture

Recovery of the desired product from the C. glutamicum cells orsupernatant of the above-described culture can be performed by variousmethods well known in the art. If the desired product is not secretedfrom the cells, the cells can be harvested from the culture by low-speedcentrifugation, the cells can be lysed by standard techniques, such asmechanical force or sonication. The cellular debris is removed bycentrifugation, and the supernatant fraction containing the solubleproteins is retained for further purification of the desired compound.If the product is secreted from the C. glutamicum cells, then the cellsare removed from the culture by low-speed centrifugation, and thesupernate fraction is retained for further purification.

The supernatant fraction from either purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on a chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by the resinwhile the sample is not. Such chromatography steps may be repeated asnecessary, using the same or different chromatography resins. One ofordinary skill in the art would be well-versed in the selection ofappropriate chromatography resins and in their most efficaciousapplication for a particular molecule to be purified. The purifiedproduct may be concentrated by filtration or ultrafiltration, and storedat a temperature at which the stability of the product is maximized.

There are a wide array of purification methods known to the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey, J. E. &Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: New York(1986).

The identity and purity of the isolated compounds may be assessed bytechniques standard in the art. These include high-performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, NIRS, enzymatic assay, or microbiologically. Suchanalysis methods are reviewed in: Patek et al. (1994) Appl. Environ.Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11:27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70.Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH:Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p.581-587; Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, vol. 17.

EXAMPLE 11 Analysis of the Gene Sequences of the Invention

The comparison of sequences and determination of percent homologybetween two sequences are art-known techniques, and can be accomplishedusing a mathematical algorithm, such as the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad Sci. USA 87:2264-68, modified as inKarlin and Altschul (1993) Proc. Natl. Acad Sci. USA 90:5873-77. Such analgorithm is incorporated into the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to PTS nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to PTS protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one ofordinary skill in the art will know how to optimize the parameters ofthe program (e.g., XBLAST and NBLAST) for the specific sequence beinganalyzed.

Another example of a mathematical algorithm utilized for the comparisonof sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl.Biosci. 4: 11-17). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art, and include ADVANCE and ADAM.described in Torelli and Robotti (1994) Comput. Appl. Biosci. 10:3-5;and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.

The percent homology between two amino acid sequences can also beaccomplished using the GAP program in the GCG software package(available at http://www.gcg.com), using either a Blosum 62 matrix or aPAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a lengthweight of 2, 3, or 4. The percent homology between two nucleic acidsequences can be accomplished using the GAP program in the GCG softwarepackage, using standard parameters, such as a gap weight of 50 and alength weight of 3.

A comparative analysis of the gene sequences of the invention with thosepresent in Genbank has been performed using techniques known in the art(see, e.g., Bexevanis and Ouellette, eds. (1998) Bioinformatics: APractical Guide to the Analysis of Genes and Proteins. John Wiley andSons: New York). The gene sequences of the invention were compared togenes present in Genbank in a three-step process. In a first step, aBLASTN analysis (e.g., a local alignment analysis) was performed foreach of the sequences of the invention against the nucleotide sequencespresent in Genbank, and the top 500 hits were retained for furtheranalysis. A subsequent FASTA search (e.g., a combined local and globalalignment analysis, in which limited regions of the sequences arealigned) was performed on these 500 hits. Each gene sequence of theinvention was subsequently globally aligned to each of the top threeFASTA hits, using the GAP program in the GCG software package (usingstandard parameters). In order to obtain correct results, the length ofthe sequences extracted from Genbank were adjusted to the length of thequery sequences by methods well-known in the art. The results of thisanalysis are set forth in Table 4. The resulting data is identical tothat which would have been obtained had a GAP (global) analysis alonebeen performed on each of the genes of the invention in comparison witheach of the references in Genbank, but required significantly reducedcomputational time as compared to such a database-wide GAP (global)analysis. Sequences of the invention for which no alignments above thecutoff values were obtained are indicated on Table 4 by the absence ofalignment information. It will further be understood by one of ordinaryskill in the art that the GAP alignment homology percentages set forthin Table 4 under the heading “% homology (GAP)” are listed in theEuropean numerical format, wherein a ‘,’ represents a decimal point. Forexample, a value of “40,345” in this column represents “40.345%”.

EXAMPLE 12 Construction and Operation of DNA Microarrays

The sequences of the invention may additionally be used in theconstruction and application of DNA microarrays (the design,methodology, and uses of DNA arrays are well known in the art, and aredescribed, for example, in Schena, M. et al. (1995) Science 270:467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367;DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi,J. L. et al. (1997) Science 278: 680-686).

DNA microarrays are solid or flexible supports consisting ofnitrocellulose, nylon, glass, silicone, or other materials. Nucleic acidmolecules may be attached to the surface in an ordered manner. Afterappropriate labeling, other nucleic acids or nucleic acid mixtures canbe hybridized to the immobilized nucleic acid molecules, and the labelmay be used to monitor and measure the individual signal intensities ofthe hybridized molecules at defined regions. This methodology allows thesimultaneous quantification of the relative or absolute amount of all orselected nucleic acids in the applied nucleic acid sample or mixture.DNA microarrays, therefore, permit an analysis of the expression ofmultiple (as many as 6800 or more) nucleic acids in parallel (see, e.g.,Schena, M. (1996) BioEssays 18(5): 427-431).

The sequences of the invention may be used to design oligonucleotideprimers which are able to amplify defined regions of one or more C.glutamicum genes by a nucleic acid amplification reaction such as thepolymerase chain reaction. The choice and design of the 5′ or 3′oligonucleotide primers or of appropriate linkers allows the covalentattachment of the resulting PCR products to the surface of a supportmedium described above (and also described, for example, Schena, M. etal. (1995) Science 270: 467-470).

Nucleic acid microarrays may also be constructed by in situoligonucleotide synthesis as described by Wodicka, L. et al. (1997)Nature Biotechnology 15: 1359-1367. By photolithographic methods,precisely defined regions of the matrix are exposed to light. Protectivegroups which are photolabile are thereby activated and undergonucleotide addition, whereas regions that are masked from light do notundergo any modification. Subsequent cycles of protection and lightactivation permit the synthesis of different oligonucleotides at definedpositions. Small, defined regions of the genes of the invention may besynthesized on microarrays by solid phase oligonucleotide synthesis.

The nucleic acid molecules of the invention present in a sample ormixture of nucleotides may be hybridized to the microarrays. Thesenucleic acid molecules can be labeled according to standard methods. Inbrief, nucleic acid molecules (e.g., mRNA molecules or DNA molecules)are labeled by the incorporation of isotopically or fluorescentlylabeled nucleotides, e.g., during reverse transcription or DNAsynthesis. Hybridization of labeled nucleic acids to microarrays isdescribed (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al.(1997), supra; and DeSaizieu A. et al. (1998), supra). The detection andquantification of the hybridized molecule are tailored to the specificincorporated label. Radioactive labels can be detected, for example, asdescribed in Schena, M. et al. (1995) supra) and fluorescent labels maybe detected, for example, by the method of Shalon et al. (1996) GenomeResearch 6: 639-645).

The application of the sequences of the invention to DNA microarraytechnology, as described above, permits comparative analyses ofdifferent strains of C. glutamicum or other Corynebacteria. For example,studies of inter-strain variations based on individual transcriptprofiles and the identification of genes that are important for specificand/or desired strain properties such as pathogenicity, productivity andstress tolerance are facilitated by nucleic acid array methodologies.Also, comparisons of the profile of expression of genes of the inventionduring the course of a fermentation reaction are possible using nucleicacid array technology.

EXAMPLE 13 Analysis of the Dynamics of Cellular Protein Populations(Proteomics)

The genes, compositions, and methods of the invention may be applied tostudy the interactions and dynamics of populations of proteins, termed‘proteomics’. Protein populations of interest include, but are notlimited to, the total protein population of C. glutamicum (e.g., incomparison with the protein populations of other organisms), thoseproteins which are active under specific environmental or metabolicconditions (e.g., during fermentation, at high or low temperature, or athigh or low pH), or those proteins which are active during specificphases of growth and development.

Protein populations can be analyzed by various well-known techniques,such as gel electrophoresis. Cellular proteins may be obtained, forexample, by lysis or extraction, and may be separated from one anotherusing a variety of electrophoretic techniques. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) separates proteins largelyon the basis of their molecular weight. Isoelectric focusingpolyacrylamide gel electrophoresis (IEF-PAGE) separates proteins bytheir isoelectric point (which reflects not only the amino acid sequencebut also posttranslational modifications of the protein). Another, morepreferred method of protein analysis is the consecutive combination ofboth IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described,for example, in Hermann et al. (1998) Electrophoresis 19: 3217-3221;Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al.(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997)Electrophoresis 18: 1451-1463). Other separation techniques may also beutilized for protein separation, such as capillary gel electrophoresis;such techniques are well known in the art.

Proteins separated by these methodologies can be visualized by standardtechniques, such as by staining or labeling. Suitable stains are knownin the art, and include Coomassie Brilliant Blue, silver stain, orfluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion ofradioactively labeled amino acids or other protein precursors (e.g.,³⁵S-methionine, ³⁵-cysteine, ¹⁴C-labelled amino acids, ¹⁵N-amino acids,¹⁵NO₃ or ¹⁵NH₄ ⁺ or ¹³C-labelled amino acids) in the medium of C.glutamicum permits the labeling of proteins from these cells prior totheir separation. Similarly, fluorescent labels may be employed. Theselabeled proteins can be extracted, isolated and separated according tothe previously described techniques.

Proteins visualized by these techniques can be further analyzed bymeasuring the amount of dye or label used. The amount of a given proteincan be determined quantitatively using, for example, optical methods andcan be compared to the amount of other proteins in the same gel or inother gels. Comparisons of proteins on gels can be made, for example, byoptical comparison, by spectroscopy, by image scanning and analysis ofgels, or through the use of photographic films and screens. Suchtechniques are well-known in the art.

To determine the identity of any given protein, direct sequencing orother standard techniques may be employed. For example, N— and/orC-terminal amino acid sequencing (such as Edman degradation) may beused, as may mass spectrometry (in particular MALDI or ESI techniques(see, e.g., Langen et al. (1997) Electrophoresis 18: 1184-1192)). Theprotein sequences provided herein can be used for the identification ofC. glutamicum proteins by these techniques.

The information obtained by these methods can be used to comparepatterns of protein presence, activity, or modification betweendifferent samples from various biological conditions (e.g., differentorganisms, time points of fermentation, media conditions, or differentbiotopes, among others). Data obtained from such experiments alone, orin combination with other techniques, can be used for variousapplications, such as to compare the behavior of various organisms in agiven (e.g., metabolic) situation, to increase the productivity ofstrains which produce fine chemicals or to increase the efficiency ofthe production of fine chemicals.

Equivalents

Those of ordinary skill in the art will recognize, or will be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.TABLE 1 Genes Included in the Invention PHOSPHOENOLPYRUVATE: SUGARPHOSPHOTRANSFERASE SYSTEM Nucle- Amino otide Acid SEQ ID SEQ IDIdentification NT NT NO NO Code Contig. Start Stop Function 1 2 RXS00315PTS SYSTEM, SUCROSE-SPECIFIC IIABC COMPONENT (EIIABC-SCR)(SUCROSE-PERMEASE IIABC COMPONENT(PHOSPHOTRANSFERASE ENZYME II, ABCCOMPONENT) (EC 2.7.1.69) 3 4 F RXA00315 GR00053 6537 5452 PTS SYSTEM,BETA-GLUCOSIDES-SPECIFIC IIABC COMPONENT (EIIABC-BGL)(BETA-GLUCOSIDES-PERMEASE IIABC COMPONENT) (PHOSPHOTRANSFERASE ENZYMEII, ABC COMPONENT) (EC 2.7.1.69) 5 6 RXA01503 GR00424 10392 10640 PTSSYSTEM, BETA-GLUCOSIDES-SPECIFIC IIABC COMPONENT (EIIABC-BGL)(BETA-GLUCOSIDES-PERMEASE IIABC COMPONENT) (PHOSPHOTRANSFERASE ENZYMEII, ABC COMPONENT) (EC 2.7.1.69) 7 8 RXN01299 VV0068 11954 9891 PTSSYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 9 10 F RXA01299GR00375 6 446 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69)11 12 F RXA01883 GR00538 2154 2633 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBCCOMPONENT (EC 2.7.1.69) 13 14 F RXA01889 GR00540 77 631 PTS SYSTEM,FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 15 16 RXA00951 GR00261564 172 PTS SYSTEM, MANNITOL (CRYPTIC)-SPECIFIC IIA COMPONENT(EIIA-(C)MTL) (MANNITOL (CRYPTIC)-PERMEASE IIA COMPONENT)(PHOSPHOTRANSFERASE ENZYME II, A COMPONENT) (EC 2.7.1.69) 17 18 RXN01244VV0068 14141 15844 PHOSPHOENOLPYRUVATE-PROTEIN PHOSPHOTRANSFERASE (EC2.7.3.9) 19 20 F RXA01244 GR00359 4837 3329 PHOSPHOENOLPYRUVATE-PROTEINPHOSPHOTRANSFERASE (EC 2.7.3.9) 21 22 RXA01300 GR00375 637 903PHOSPHOCARRIER PROTEIN HPR 23 24 RXN03002 VV0236 1437 1844 PTS SYSTEM,MANNITOL (CRYPTIC)-SPECIFIC IIA COMPONENT (EIIA-(C)MTL) (MANNITOL(CRYPTIC)-PERMEASE IIA COMPONENT) (PHOSPHOTRANSFERASE ENZYME II, ACOMPONENT) (EC 2.7.1.69) 25 26 RXC00953 VV0260 1834 1082 MembraneSpanning Protein involved in PTS system 27 28 RXC03001 Membrane SpanningProtein involved in PTS system 29 30 RXN01943 VV0120 4326 6374 PTSSYSTEM, GLUCOSE-SPECIFIC IIABC COMPONENT (EC 2.7.1.69) 31 32 F RXA02191GR00642 3395 4633 PHOSPHOENOLPYRUVATE SUGAR PHOSPHOTRANSFERASE 33 34 FRXA01943 GR00557 3944 3540 crr gene; phosphotransferase systemglucose-specific enzyme III

TABLE 2 GENES IDENTIFIED FROM GENBANK GenBank ™ Accession No. Gene NameGene Function Reference A09073 ppg Phosphoenol pyruvate carboxylaseBachmann, B. et al. “DNA fragment coding for phosphoenolpyruvatcorboxylase, recombinant DNA carrying said fragment, strains carryingthe recombinant DNA and method for producing L-aminino acids using saidstrains,” Patent: EP 0358940-A 3 Mar. 21, 1990 A45579, Threoninedehydratase Moeckel, B. et al. “Production of L-isoleucine by means ofrecombinant A45581, micro-organisms with deregulated threoninedehydratase,” Patent: WO A45583, 9519442-A 5 Jul. 20, 1995 A45585 A45587AB003132 murC; ftsQ; ftsZ Kobayashi, M. et al. “Cloning, sequencing, andcharacterization of the ftsZ gene from coryneform bacteria,” Biochem.Biophys. Res. Commun., 236(2): 383-388 (1997) AB015023 murC; ftsQ Wachi,M. et al. “A murC gene from Coryneform bacteria,” Appl. Microbiol.Biotechnol., 51(2): 223-228 (1999) AB018530 dtsR Kimura, E. et al.“Molecular cloning of a novel gene, dtsR, which rescues the detergentsensitivity of a mutant derived from Brevibacterium lactofermentum,”Biosci. Biotechnol. Biochem., 60(10): 1565-1570 (1996) AB018531 dtsR1;dtsR2 AB020624 murI D-glutamate racemase AB023377 tkt transketolaseAB024708 gltB; gltD Glutamine 2-oxoglutarate aminotransferase large andsmall subunits AB025424 acn aconitase AB027714 rep Replication proteinAB027715 rep; and Replication protein; aminoglycoside adenyltransferaseAF005242 argC N-acetylglutamate-5-semialdehyde dehydrogenase AF005635glnA Glutamine synthetase AF030405 hisF cyclase AF030520 argGArgininosuccinate synthetase AF031518 argF Ornithinecarbamolytransferase AF036932 aroD 3-dehydroquinate dehydratase AF038548pyc Pyruvate carboxylase AF038651 dciAE; apt; rel Dipeptide-bindingprotein; adenine Wehmeier, L. et al. “The role of the Corynebacteriumglutamicum rel gene in phosphoribosyltransferase; GTP (p)ppGppmetabolism,” Microbiology, 144: 1853-1862 (1998) pyrophosphokinaseAF041436 argR Arginine repressor AF045998 impA Inositol monophosphatephosphatase AF048764 argH Argininosuccinate lyase AF049897 argC; argJ;argB; N-acetylglutamylphosphate reductase; argD; argF; argR; ornithineacetyltransferase; N- argG; argH acetylglutamate kinase; acetylornithinetransminase; ornithine carbamoyltransferase; arginine repressor;argininosuccinate synthase; argininosuccinate lyase AF050109 inhAEnoyl-acyl carrier protein reductase AF050166 hisG ATPphosphoribosyltransferase AF051846 hisAPhosphoribosylformimino-5-amino-1- phosphoribosyl-4-imidazolecarboxamideisomerase AF052652 metA Homoserine O-acetyltransferase Park, S. et al.“Isolation and analysis of metA, a methionine biosynthetic gene encodinghomoserine acetyltransferase in Corynebacterium glutamicum,” Mol.Cells., 8(3): 286-294 (1998) AF053071 aroB Dehydroquinate synthetaseAF060558 hisH Glutamine amidotransferase AF086704 hisEPhosphoribosyl-ATP- pyrophosphohydrolase AF114233 aroA5-enolpyruvylshikimate 3-phosphate synthase AF116184 panDL-aspartate-alpha-decarboxylase precursor Dusch, N. et al. “Expressionof the Corynebacterium glutamicum panD gene encodingL-aspartate-alpha-decarboxylase leads to pantothenate overproduction inEscherichia coli,” Appl. Environ. Microbiol., 65(4)1530-1539 (1999)AF124518 aroD; aroE 3-dehydroquinase; shikimate dehydrogenase AF124600aroC; aroK; aroB; Chorismate synthase; shikimate kinase; 3- pepQdehydroquinate synthase; putative cytoplasmic peptidase AF145897 inhAAF145898 inhA AJ001436 ectP Transport of ectoine, glycine betaine,Peter, H. et al. “Corynebacterium glutamicum is equipped with foursecondary proline carriers for compatible solutes: Identification,sequencing, and characterization of the proline/ectoine uptake system,ProP, and the ectoine/proline/glycine betaine carrier, EctP,” J.Bacteriol., 180(22): 6005-6012 (1998) AJ004934 dapDTetrahydrodipicolinate succinylase Wehrmann, A. et al. “Different modesof diaminopimelate synthesis and their (incomplete^(i)) role in cellwall integrity: A study with Corynebacterium glutamicum,” J. Bacteriol.,180(12): 3159-3165 (1998) AJ007732 ppc; secG; amt; ocd;Phosphoenolpyruvate-carboxylase; ?; high soxA affinity ammonium uptakeprotein; putative ornithine-cyclodecarboxylase; sarcosine oxidaseAJ010319 ftsY, glnB, glnD; srp; Involved in cell division; PII protein;Jakoby, M. et al. “Nitrogen regulation in Corynebacterium glutamicum;amtP uridylyltransferase (uridylyl-removing Isolation of genes involvedin biochemical characterization of corresponding enzmye); signalrecognition particle; low proteins,” FEMS Microbiol., 173(2): 303-310(1999) affinity ammonium uptake protein AJ132968 cat Chloramphenicolaceteyl transferase AJ224946 mqo L-malate: quinone oxidoreductaseMolenaar, D. et al. “Biochemical and genetic characterization of themembrane-associated malate dehydrogenase (acceptor) from Corynebacteriumglutamicum,” Eur. J. Biochem., 254(2): 395-403 (1998) AJ238250 ndh NADHdehydrogenase AJ238703 porA Porin Lichtinger, T. et al. “Biochemical andbiophysical characterization of the cell wall porin of Corynebacteriumglutamicum: The channel is formed by a low molecular mass polypeptide,”Biochemistry, 37(43): 15024-15032 (1998) D17429 Transposable elementIS31831 Vertes, A. A. et al. “Isolation and characterization of IS31831,a transposable element from Corynebacterium glutamicum,” Mol.Microbiol., 11(4): 739-746 (1994) D84102 odhA 2-oxoglutaratedehydrogenase Usuda, Y. et al. “Molecular cloning of the Corynebacteriumglutamicum (Brevibacterium lactofermentum AJ12036) odhA gene encoding anovel type of 2-oxoglutarate dehydrogenase,” Microbiology, 142:3347-3354 (1996) E01358 hdh; hk Homoserine dehydrogenase; homoserineKatsumata, R. et al. “Production of L-thereonine and L-isoleucine,”Patent: JP kinase 1987232392-A 1 Oct. 12, 1987 E01359 Upstream of thestart codon of homoserine Katsumata, R. et al. “Production ofL-thereonine and L-isoleucine,” Patent: JP kinase gene 1987232392-A 2Oct. 12, 1987 E01375 Tryptophan operon E01376 trpL; trpE Leader peptide;anthranilate synthase Matsui, K. et al. “Tryptophan operon, peptide andprotein coded thereby, utilization of tryptophan operon gene expressionand production of tryptophan,” Patent: JP 1987244382-A 1 Oct. 24, 1987E01377 Promoter and operator regions of Matsui, K. et al. “Tryptophanoperon, peptide and protein coded thereby, tryptophan operon utilizationof tryptophan operon gene expression and production of tryptophan,”Patent: JP 1987244382-A 1 Oct. 24, 1987 E03937 Biotin-synthaseHatakeyama, K. et al. “DNA fragment containing gene capable of codingbiotin synthetase and its utilization,” Patent: JP 1992278088-A 1 Oct.02, 1992 E04040 Diamino pelargonic acid aminotransferase Kohama, K. etal. “Gene coding diaminopelargonic acid aminotransferase anddesthiobiotin synthetase and its utilization,” Patent: JP 1992330284-A 1Nov. 18, 1992 E04041 Desthiobiotinsynthetase Kohama, K. et al. “Genecoding diaminopelargonic acid aminotransferase and desthiobiotinsynthetase and its utilization,” Patent: JP 1992330284-A 1 Nov. 18, 1992E04307 Flavum aspartase Kurusu, Y. et al. “Gene DNA coding aspartase andutilization thereof,” Patent: JP 1993030977-A 1 Feb. 09, 1993 E04376Isocitric acid lyase Katsumata, R. et al. “Gene manifestationcontrolling DNA,” Patent: JP 1993056782-A 3 Mar. 09, 1993 E04377Isocitric acid lyase N-terminal fragment Katsumata, R. et al. “Genemanifestation controlling DNA,” Patent: JP 1993056782-A 3 Mar. 09, 1993E04484 Prephenate dehydratase Sotouchi, N. et al. “Production ofL-phenylalanine by fermentation,” Patent: JP 1993076352-A 2 Mar. 30,1993 E05108 Aspartokinase Fugono, N. et al. “Gene DNA codingAspartokinase and its use,” Patent: JP 1993184366-A 1 Jul. 27, 1993E05112 Dihydro-dipichorinate synthetase Hatakeyama, K. et al. “Gene DNAcoding dihydrodipicolinic acid synthetase and its use,” Patent: JP1993184371-A 1 Jul. 27, 1993 E05776 Diaminopimelic acid dehydrogenaseKobayashi, M. et al. “Gene DNA coding Diaminopimelic acid dehydrogenaseand its use,” Patent: JP 1993284970-A 1 Nov. 02, 1993 E05779 Threoninesynthase Kohama, K. et al. “Gene DNA coding threonine synthase and itsuse,” Patent: JP 1993284972-A 1 Nov. 02, 1993 E06110 Prephenatedehydratase Kikuchi, T. et al. “Production of L-phenylalanine byfermentation method,” Patent: JP 1993344881-A 1 Dec. 27, 1993 E06111Mutated Prephenate dehydratase Kikuchi, T. et al. “Production ofL-phenylalanine by fermentation method,” Patent: JP 1993344881-A 1 Dec.27, 1993 E06146 Acetohydroxy acid synthetase Inui, M. et al. “Genecapable of coding Acetohydroxy acid synthetase and its use,” Patent: JP1993344893-A 1 Dec. 27, 1993 E06825 Aspartokinase Sugimoto, M. et al.“Mutant aspartokinase gene,” patent: JP 1994062866-A 1 Mar. 08, 1994E06826 Mutated aspartokinase alpha subunit Sugimoto, M. et al. “Mutantaspartokinase gene,” patent: JP 1994062866-A 1 Mar. 08, 1994 E06827Mutated aspartokinase alpha subunit Sugimoto, M. et al. “Mutantaspartokinase gene,” patent: JP 1994062866-A 1 Mar. 08, 1994 E07701 secYHonno, N. et al. “Gene DNA participating in integration of membraneousprotein to membrane,” Patent: JP 1994169780-A 1 Jun. 21, 1994 E08177Aspartokinase Sato, Y. et al. “Genetic DNA capable of codingAspartokinase released from feedback inhibition and its utilization,”Patent: JP 1994261766-A 1 Sep. 20, 1994 E08178, Feedbackinhibition-released Aspartokinase Sato, Y. et al. “Genetic DNA capableof coding Aspartokinase released from E08179, feedback inhibition andits utilization,” Patent: JP 1994261766-A 1 Sep. 20, 1994 E08180,E08181, E08182 E08232 Acetohydroxy-acid isomeroreductase Inui, M. et al.“Gene DNA coding acetohydroxy acid isomeroreductase,” Patent: JP1994277067-A 1 Oct. 04, 1994 E08234 secE Asai, Y. et al. “Gene DNAcoding for translocation machinery of protein,” Patent: JP 1994277073-A1 Oct. 04, 1994 E08643 FT aminotransferase and desthiobiotin Hatakeyama,K. et al. “DNA fragment having promoter function in synthetase promoterregion coryneform bacterium,” Patent: JP 1995031476-A 1 Feb. 03, 1995E08646 Biotin synthetase Hatakeyama, K. et al. “DNA fragment havingpromoter function in coryneform bacterium,” Patent: JP 1995031476-A 1Feb. 03, 1995 E08649 Aspartase Kohama, K. et al “DNA fragment havingpromoter function in coryneform bacterium,” Patent: JP 1995031478-A 1Feb. 03, 1995 E08900 Dihydrodipicolinate reductase Madori, M. et al.“DNA fragment containing gene coding Dihydrodipicolinate acid reductaseand utilization thereof,” Patent: JP 1995075578-A 1 Mar. 20, 1995 E08901Diaminopimelic acid decarboxylase Madori, M. et al. “DNA fragmentcontaining gene coding Diaminopimelic acid decarboxylase and utilizationthereof,” Patent: JP 1995075579-A 1 Mar. 20, 95 E12594 Serinehydroxymethyltransferase Hatakeyama, K. et al. “Production ofL-trypophan,” Patent: JP 1997028391-A 1 Feb. 04, 1997 E12760,transposase Moriya, M. et al. “Amplification of gene using artificialtransposon,” Patent: E12759, JP 1997070291-A Mar. 18, 1997 E12758 E12764Arginyl-tRNA synthetase; diaminopimelic Moriya, M. et al. “Amplificationof gene using artificial transposon,” Patent: acid decarboxylase JP1997070291-A Mar. 18, 1997 E12767 Dihydrodipicolinic acid synthetaseMoriya, M. et al. “Amplification of gene using artificial transposon,”Patent: JP 1997070291-A Mar. 18, 1997 E12770 aspartokinase Moriya, M. etal. “Amplification of gene using artificial transposon,” Patent: JP1997070291-A Mar. 18, 1997 E12773 Dihydrodipicolinic acid reductaseMoriya, M. et al. “Amplification of gene using artificial transposon,”Patent: JP 1997070291-A Mar. 18, 1997 E13655 Glucose-6-phosphatedehydrogenase Hatakeyama, K. et al. “Glucose-6-phosphate dehydrogenaseand DNA capable of coding the same,” Patent: JP 1997224661-A 1 Sep. 02,1997 L01508 IlvA Threonine dehydratase Moeckel, B. et al. “Functionaland structural analysis of the threonine dehydratase of Corynebacteriumglutamicum,” J. Bacteriol., 174: 8065-8072 (1992) L07603 EC 4.2.1.153-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. “The cloning andnucleotide sequence of Corynebacterium phosphate synthase glutamicum3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene,” FEMSMicrobiol. Lett., 107: 223-230 (1993) L09232 IlvB; ilvN; ilvCAcetohydroxy acid synthase large subunit; Keilhauer, C. et al.“Isoleucine synthesis in Corynebacterium glutamicum: Acetohydroxy acidsynthase small subunit; molecular analysis of the ilvB-ilvN-ilvCoperon,” J. Bacteriol., 175(17): 5595-5603 Acetohydroxy acidisomeroreductase (1993) L18874 PtsM Phosphoenolpyruvate sugar Fouet, Aet al. “Bacillus subtilis sucrose-specific enzyme II of thephosphotransferase phosphotransferase system: expression in Escherichiacoli and homology to enzymes II from enteric bacteria,” PNAS USA,84(24): 8773-8777 (1987); Lee, J. K. et al. “Nucleotide sequence of thegene encoding the Corynebacterium glutamicum mannose enzyme II andanalyses of the deduced protein sequence,” FEMS Microbiol. Lett.,119(1-2): 137-145 (1994) L27123 aceB Malate synthase Lee, H-S. et al.“Molecular characterization of aceB, a gene encoding malate synthase inCorynebacterium glutamicum,” J. Microbiol. Biotechnol., 4(4): 256-263(1994) L27126 Pyruvate kinase Jetten, M. S. et al. “Structural andfunctional analysis of pyruvate kinase from Corynebacterium glutamicum,”Appl. Environ. Microbiol., 60(7): 2501-2507 (1994) L28760 aceAIsocitrate lyase L35906 dtxr Diphtheria toxin repressor Oguiza, J. A. etal. “Molecular cloning, DNA sequence analysis, and characterization ofthe Corynebacterium diphtheriae dtxR from Brevibacteriumlactofermentum,” J. Bacteriol., 177(2): 465-467 (1995) M13774 Prephenatedehydratase Follettie, M. T. et al. “Molecular cloning and nucleotidesequence of the Corynebacterium glutamicum pheA gene,” J. Bacteriol.,167: 695-702 (1986) M16175 5S rRNA Park, Y-H. et al. “Phylogeneticanalysis of the coryneform bacteria by 56 rRNA sequences,” J.Bacteriol., 169: 1801-1806 (1987) M16663 trpE Anthranilate synthase, 5′end Sano, K. et al. “Structure and function of the trp operon controlregions of Brevibacterium lactofermentum, a glutamic-acid-producingbacterium,” Gene, 52: 191-200 (1987) M16664 trpA Tryptophan synthase,3′end Sano, K. et al. “Structure and function of the trp operon controlregions of Brevibacterium lactofermentum, a glutamic-acid-producingbacterium,” Gene, 52: 191-200 (1987) M25819 Phosphoenolpyruvatecarboxylase O'Regan, M. et al. “Cloning and nucleotide sequence of thePhosphoenolpyruvate carboxylase-coding gene of Corynebacteriumglutamicum ATCC13032,” Gene, 77(2): 237-251 (1989) M85106 23S rRNA geneinsertion sequence Roller, C. et al. “Gram-positive bacteria with a highDNA G + C content are characterized by a common insertion within their23S rRNA genes,” J. Gen. Microbiol., 138: 1167-1175 (1992) M85107, 23SrRNA gene insertion sequence Roller, C. et al. “Gram-positive bacteriawith a high DNA G + C content are M85108 characterized by a commoninsertion within their 23S rRNA genes,” J. Gen. Microbiol., 138:1167-1175 (1992) M89931 aecD; brnQ; yhbw Beta C-S lyase; branched-chainamino acid uptake carrier; hypothetical Rossol, I. et al. “TheCorynebacterium glutamicum aecD gene encodes a C-S protein yhbw lyasewith alpha, beta-elimination activity that degrades aminoethylcysteine,”J. Bacteriol., 174(9): 2968-2977 (1992); Tauch, A. et al. “Isoleucineuptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQgene product,” Arch. Microbiol., 169(4): 303-312 (1998) S59299 trpLeader gene (promoter) Herry, D. M. et al. “Cloning of the trp genecluster from a tryptophan- hyperproducing strain of Corynebacteriumglutamicum: identification of a mutation in the trp leader sequence,”Appl. Environ. Microbiol., 59(3): 791-799 (1993) U11545 trpDAnthranilate phosphoribosyltransferase O'Gara, J. P. and Dunican, L. K.(1994) Complete nucleotide sequence of the Corynebacterium glutamicumATCC 21850 tpD gene.” Thesis, Microbiology Department, UniversityCollege Galway, Ireland. U13922 cglIM; cglIR; clgIIR Putative type II5-cytosoine Schafer, A. et al. “Cloning and characterization of a DNAregion encoding a methyltransferase; putative type II stress-sensitiverestriction system from Corynebacterium glutamicum ATCC restrictionendonuclease; putative type I or 13032 and analysis of its role inintergeneric conjugation with Escherichia type III restrictionendonuclease coli,” J. Bacteriol., 176(23): 7309-7319 (1994); Schafer,A. et al. “The Corynebacterium glutamicum cglIM gene encoding a5-cytosine in an McrBC- deficient Escherichia coli strain,” Gene,203(2): 95-101 (1997) U14965 recA U31224 ppx Ankri, S. et al. “Mutationsin the Corynebacterium glutamicumproline biosynthetic pathway: A naturalbypass of the proA step,” J. Bacteriol., 178(15): 4412-4419 (1996)U31225 proC L-proline: NADP+ 5-oxidoreductase Ankri, S. et al.“Mutations in the Corynebacterium glutamicumproline biosyntheticpathway: A natural bypass of the proA step,” J. Bacteriol., 178(15):4412-4419 (1996) U31230 obg; proB; unkdh ?; gamma glutamyl kinase;similar to D- Ankri, S. et al. “Mutations in the Corynebacteriumglutamicumproline isomer specific 2-hydroxyacid biosynthetic pathway: Anatural bypass of the proA step,” J. Bacteriol., dehydrogenases 178(15):4412-4419 (1996) U31281 bioB Biotin synthase Serebriiskii, I. G., “Twonew members of the bio B superfamily: Cloning, sequencing and expressionof bio B genes of Methylobacillus flagellatum and Corynebacteriumglutamicum,” Gene, 175: 15-22 (1996) U35023 thtR; accBC Thiosulfatesulfurtransferase; acyl CoA Jager, W. et al. “A Corynebacteriumglutamicum gene encoding a two-domain carboxylase protein similar tobiotin carboxylases and biotin-carboxyl-carrier proteins,” Arch.Microbiol., 166(2); 76-82 (1996) U43535 cmr Multidrug resistance proteinJager, W. et al. “A Corynebacterium glutamicum gene conferring multidrugresistance in the heterologous host Escherichia coli,” J. Bacteriol.,179(7): 2449-2451 (1997) U43536 clpB Heat shock ATP-binding proteinU53587 aphA-3 3′5″-aminoglycoside phosphotransferase U89648Corynebacterium glutamicum unidentified sequence involved in histidinebiosynthesis, partial sequence X04960 trpA; trpB; trpC; trpD; Tryptophanoperon Matsui, K. et al. “Complete nucleotide and deduced amino acidsequences of trpE; trpG; trpL the Brevibacterium lactofermentumtryptophan operon,” Nucleic Acids Res., 14(24): 10113-10114 (1986)X07563 lys A DAP decarboxylase (meso-diaminopimelate Yeh, P. et al.“Nucleic sequence of the lysA gene of Corynebacterium decarboxylase, EC4.1.1.20) glutamicum and possible mechanisms for modulation of itsexpression,” Mol. Gen. Genet., 212(1): 112-119 (1988) X14234 EC 4.1.1.31Phosphoenolpyruvate carboxylase Eikmanns, B. J. et al. “ThePhosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum:Molecular cloning, nucleotide sequence, and expression,” Mol. Gen.Genet., 218(2): 330-339 (1989); Lepiniec, L. et al. “SorghumPhosphoenolpyruvate carboxylase gene family: structure, function andmolecular evolution,” Plant. Mol. Biol., 21 (3): 487-502 (1993) X17313fda Fructose-bisphosphate aldolase Von der Osten, C. H. et al.“Molecular cloning, nucleotide sequence and fine- structural analysis ofthe Corynebacterium glutamicum fda gene: structural comparison of C.glutamicum fructose-1,6-biphosphate aldolase to class I and class IIaldolases,” Mol. Microbiol., X53993 dapA L-2,3-dihydrodipicolinatesynthetase (EC Bonnassie, S. et al. “Nucleic sequence of the dapA genefrom 4.2.1.52) Corynebacterium glutamicum,” Nucleic Acids Res., 18(21):6421 (1990) X54223 AttB-related site Cianciotto, N. et al. “DNA sequencehomology between att B-related sites of Corynebacterium diphtheriae,Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP siteof lambdacorynephage,” FEMS. Microbiol, Lett., 66: 299-302 (1990) X54740argS; lysA Arginyl-tRNA synthetase; Diaminopimelate Marcel, T. et al.“Nucleotide sequence and organization of the upstream regiondecarboxylase of the Corynebacterium glutamicum lysA gene,” Mol.Microbiol., 4(11): 1819-1830 (1990) X55994 trpL; trpE Putative leaderpeptide; anthranilate Heery, D. M. et al. “Nucleotide sequence of theCorynebacterium glutamicum synthase component 1 trpE gene,” NucleicAcids Res., 18(23): 7138 (1990) X56037 thrC Threonine synthase Han, K.S. et al. “The molecular structure of the Corynebacterium glutamicumthreonine synthase gene,” Mol. Microbiol., 4(10): 1693-1702 (1990)X56075 attB-related site Attachment site Cianciotto, N. et al. “DNAsequence homology between att B-related sites of Corynebacteriumdiphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, andthe attP site of lambdacorynephage,” FEMS. Microbiol, Lett., 66: 299-302(1990) X57226 lysC-alpha; lysC-beta; Aspartokinase-alpha subunit;Kalinowski, J. et al. “Genetic and biochemical analysis of theAspartokinase asd Aspartokinase-beta subunit; aspartate beta fromCorynebacterium glutamicum,” Mol. Microbiol., 5(5): 1197-1204 (1991);semialdehyde dehydrogenase Kalinowski, J. et al. “Aspartokinase geneslysC alpha and lysC beta overlap and are adjacent to the aspertatebeta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum,”Mol. Gen. Genet., 224(3): 317-324 (1990) X59403 gap; pgk; tpiGlyceraldehyde-3-phosphate; Eikmanns, B. J. “Identification, sequenceanalysis, and expression of a phosphoglycerate kinase; triosephosphateCorynebacterium glutamicum gene cluster encoding the three glycolyticisomerase enzymes glyceraldehyde-3-phosphate dehydrogenase,3-phosphoglycerate kinase, and triosephosphate isomeras,” J. Bacteriol.,174(19): 6076-6086 (1992) X59404 gdh Glutamate dehydrogenase Bormann, E.R. et al. “Molecular analysis of the Corynebacterium glutamicum gdh geneencoding glutamate dehydrogenase,” Mol. Microbiol., 6(3): 317-326 (1992)X60312 lysl L-lysine permease Seep-Feldhaus, A. H. et al. “Molecularanalysis of the Corynebacterium glutamicum lysl gene involved in lysineuptake,” Mol. Microbiol., 5(12): 2995-3005 (1991) X66078 cop1 Ps1protein Joliff, G. et al. “Cloning and nucleotide sequence of the csplgene encoding PS1, one of the two major secreted proteins ofCorynebacterium glutamicum: The deduced N-terminal region of PS1 issimilar to the Mycobacterium antigen 85 complex,” Mol. Microbiol.,6(16): 2349-2362 (1992) X66112 glt Citrate synthase Eikmanns, B. J. etal. “Cloning sequence, expression and transcriptional analysis of theCorynebacterium glutamicum gltA gene encoding citrate synthase,”Microbiol., 140: 1817-1828 (1994) X67737 dapB Dihydrodipicolinatereductase X69103 csp2 Surface layer protein PS2 Peyret, J. L. et al.“Characterization of the cspB gene encoding PS2, an orderedsurface-layer protein in Corynebacterium glutamicum,” Mol. Microbiol.,9(1): 97-109 (1993) X69104 IS3 related insertion element Bonamy, C. etal. “Identification of IS1206, a Corynebacterium glutamicum IS3-relatedinsertion sequence and phylogenetic analysis,” Mol. Microbiol., 14(3):571-581 (1994) X70959 leuA Isopropylmalate synthase Patek, M. et al.“Leucine synthesis in Corynebacterium glutamicum: enzyme activities,structure of leuA, and effect of leuA inactivation on lysine synthesis,”Appl. Environ. Microbiol., 60(1): 133-140 (1994) X71489 icd Isocitratedehydrogenase (NADP+) Eikmanns, B. J. et al. “Cloning sequence analysis,expression, and inactivation of the Corynebacterium glutamicum icd geneencoding isocitrate dehydrogenase and biochemical characterization ofthe enzyme,” J. Bacteriol., 177(3): 774-782 (1995) X72855 GDHA Glutamatedehydrogenase (NADP+) X75083, mtrA 5-methyltryptophan resistance Heery,D. M. et al. “A sequence from a tryptophan-hyperproducing strain ofX70584 Corynebacterium glutamicum encoding resistance to5-methyltryptophan,” Biochem. Biophys. Res. Commun., 201(3): 1255-1262(1994) X75085 recA Fitzpatrick, R. et al. “Construction andcharacterization of recA mutant strains of Corynebacterium glutamicumand Brevibacterium lactofermentum,” Appl. Microbiol. Biotechnol., 42(4):575-580 (1994) X75504 aceA; thiX Partial Isocitrate lyase; ? Reinscheid,D. J. et al. “Characterization of the isocitrate lyase gene fromCorynebacterium glutamicum and biochemical analysis of the enzyme,” J.Bacteriol., 176(12): 3474-3483 (1994) X76875 ATPase beta-subunit Ludwig,W. et al. “Phylogenetic relationships of bacteria based on comparativesequence analysis of elongation factor Tu and ATP-synthase beta-subunitgenes,” Antonie Van Leeuwenhoek, 64: 285-305 (1993) X77034 tufElongation factor Tu Ludwig, W. et al. “Phylogenetic relationships ofbacteria based on comparative sequence analysis of elongation factor Tuand ATP-synthase beta-subunit genes,” Antonie Van Leeuwenhoek, 64:285-305 (1993) X77384 recA Billman-Jacobe, H. “Nucleotide sequence of arecA gene from Corynebacterium glutamicum,” DNA Seq., 4(6): 403-404(1994) X78491 aceB Malate synthase Reinscheid, D. J. et al. “Malatesynthase from Corynebacterium glutamicum pta-ack operon encodingphosphotransacetylase: sequence analysis,” Microbiology, 140: 3099-3108(1994) X80629 16S rDNA 16S ribosomal RNA Rainey, F. A. et al.“Phylogenetic analysis of the genera Rhodococcus and Norcardia andevidence for the evolutionary origin of the genus Norcardia from withinthe radiation of Rhodococcus species,” Microbiol., 141: 523-528 (1995)X81191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, W. et al.“Structure of the gluABCD cluster encoding the gluD glutamate uptakesystem of Corynebacterium glutamicum,” J. Bacteriol., 177(5): 1152-1158(1995) X81379 dapE Succinyldiaminopimelate desuccinylase Wehrmann, A. etal. “Analysis of different DNA fragments of Corynebacterium glutamicumcomplementing dapE of Escherichia coli,” Microbiology, 40: 3349-56(1994) X82061 16S rDNA 16S ribosomal RNA Ruimy, R. et al. “Phylogeny ofthe genus Corynebacterium deduced from analyses of small-subunitribosomal DNA sequences,” Int. J. Syst. Bacteriol., 45(4): 740-746(1995) X82928 asd; lysC Aspartate-semialdehyde dehydrogenase; ?Serebrijski, I. et al. “Multicopy suppression by asd gene and osmoticstress- dependent complementation by heterologous proA in proA mutants,”J. Bacteriol., 177(24): 7255-7260 (1995) X82929 proA Gamma-glutamylphosphate reductase Serebrijski, I. et al. “Multicopy suppression by asdgene and osmotic stress- dependent complementation by heterologous proAin proA mutants,” J. Bacteriol., 177(24): 7255-7260 (1995) X84257 16SrDNA 16S ribosomal RNA Pascual, C. et al. “Phylogenetic analysis of thegenus Corynebacterium based on 16S rRNA gene sequences,” Int. J. Syst.Bacteriol., 45(4): 724-728 (1995) X85965 aroP; dapE Aromatic amino acidpermease; ? Wehrmann, A. et al. “Functional analysis of sequencesadjacent to dapE of Corynebacterium glutamicumproline reveals thepresence of aroP, which encodes the aromatic amino acid transporter,” J.Bacteriol., 177(20): 5991-5993 (1995) X86157 argB; argC; argD;Acetylglutamate kinase; N-acetyl-gamma- Sakanyan, V. et al. “Genes andenzymes of the acetyl cycle of arginine argF; argJ glutamyl-phosphatereductase; biosynthesis in Corynebacterium glutamicum: enzyme evolutionin the early acetylornithine aminotransferase; ornithine steps of thearginine pathway,” Microbiology, 142: 99-108 (1996)carbamoyltransferase; glutamate N- acetyltransferase X89084 pta; ackAPhosphate acetyltransferase; acetate kinase Reinscheid, D. J. et al.“Cloning, sequence analysis, expression and inactivation of theCorynebacterium glutamicum pta-ack operon encoding phosphotransacetylaseand acetate kinase,” Microbiology, 145: 503-513 (1999) X89850 attBAttachment site Le Marrec, C. et al. “Genetic characterization ofsite-specific integration functions of phi AAU2 infecting “Arthrobacteraureus C70,” J. Bacteriol., 178(7): 1996-2004 (1996) X90356 Promoterfragment F1 Patek, M. et al. “Promoters from Corynebacterium glutamicum:cloning, molecular analysis and search for a consensus motif,”Microbiology, 142: 1297-1309 (1996) X90357 Promoter fragment F2 Patek,M. et al. “Promoters from Corynebacterium glutamicum: cloning, molecularanalysis and search for a consensus motif,” Microbiology, 142: 1297-1309(1996) X90358 Promoter fragment F10 Patek, M. et al. “Promoters fromCorynebacterium glutamicum: cloning, molecular analysis and search for aconsensus motif,” Microbiology, 142: 1297-1309 (1996) X90359 Promoterfragment F13 Patek, M. et al. “Promoters from Corynebacteriumglutamicum: cloning, molecular analysis and search for a consensusmotif,” Microbiology, 142: 1297-1309 (1996) X90360 Promoter fragment F22Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,molecular analysis and search for a consensus motif,” Microbiology, 142:1297-1309 (1996) X90361 Promoter fragment F34 Patek, M. et al.“Promoters from Corynebacterium glutamicum: cloning, molecular analysisand search for a consensus motif,” Microbiology, 142: 1297-1309 (1996)X90362 Promoter fragment F37 Patek, M. et al. “Promoters fromCorynebacterium glutamicum: cloning, molecular analysis and search for aconsensus motif,” Microbiology, 142: 1297-1309 (1996) X90363 Promoterfragment F45 Patek, M. et al. “Promoters from Corynebacteriumglutamicum: cloning, molecular analysis and search for a consensusmotif,” Microbiology, 142: 1297-1309 (1996) X90364 Promoter fragment F64Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,molecular analysis and search for a consensus motif,” Microbiology, 142:1297-1309 (1996) X90365 Promoter fragment F75 Patek, M. et al.“Promoters from Corynebacterium glutamicum: cloning, molecular analysisand search for a consensus motif,” Microbiology, 142: 1297-1309 (1996)X90366 Promoter fragment PF101 Patek, M. et al. “Promoters fromCorynebacterium glutamicum: cloning, molecular analysis and search for aconsensus motif,” Microbiology, 142: 1297-1309 (1996) X90367 Promoterfragment PF104 Patek, M. et al. “Promoters from Corynebacteriumglutamicum: cloning, molecular analysis and search for a consensusmotif,” Microbiology, 142: 1297-1309 (1996) X90368 Promoter fragmentPF109 Patek, M. et al. “Promoters from Corynebacterium glutamicum:cloning, molecular analysis and search for a consensus motif,”Microbiology, 142: 1297-1309 (1996) X93513 amt Ammonium transport systemSiewe, R. M. et al. “Functional and genetic characterization of the(methyl) ammonium uptake carrier of Corynebacterium glutamicum,” J.Biol. Chem., 271(10): 5398-5403 (1996) X93514 betP Glycine betainetransport system Peter, H. et al. “Isolation, characterization, andexpression of the Corynebacterium glutamicum betP gene, encoding thetransport system for the compatible solute glycine betaine,” J.Bacteriol., 178(17): 5229-5234 (1996) X95649 orf4 Patek, M. et al.“Identification and transcriptional analysis of the dapB-ORF2- dapA-ORF4operon of Corynebacterium glutamicum, encoding two enzymes involved inL-lysine synthesis,” Biotechnol. Lett., 19: 1113-1117 (1997) X96471lysE; lysG Lysine exporter protein; Lysine export Vrljic, M. et al. “Anew type of transporter with a new type of cellular regulator proteinfunction: L-lysine export from Corynebacterium glutamicum,” Mol.Microbiol., 22(5): 815-826 (1996) X96580 panB; panC; xylB3-methyl-2-oxobutanoate Sahm, H. et al. “D-pantothenate synthesis inCorynebacterium glutamicum and hydroxymethyltransferase; pantoate-beta-use of panBC and genes encoding L-valine synthesis for D-pantothenatealanine ligase; xylulokinase overproduction,” Appl. Environ. Microbiol.,65(5): 1973-1979 (1999) X96962 Insertion sequence IS1207 and transposaseX99289 Elongation factor P Ramos, A. et al. “Cloning, sequencing andexpression of the gene encoding elongation factor P in the amino-acidproducer Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC13869),” Gene, 198: 217-222 (1997) Y00140 thrB Homoserine kinase Mateos,L. M. et al. “Nucleotide sequence of the homoserine kinase (thrB) geneof the Brevibacterium lactofermentum,” Nucleic Acids Res., 15(9): 3922(1987) Y00151 ddh Meso-diaminopimelate D-dehydrogenase Ishino, S. et al.“Nucleotide sequence of the meso-diaminopimelate D- (EC 1.4.1.16)dehydrogenase gene from Corynebacterium glutamicum,” Nucleic Acids Res.,15(9): 3917 (1987) Y00476 thrA Homoserine dehydrogenase Mateos, L. M. etal. “Nucleotide sequence of the homoserine dehydrogenase (thrA) gene ofthe Brevibacterium lactofermentum,” Nucleic Acids Res., 15(24): 10598(1987) Y00546 hom; thrB Homoserine dehydrogenase; homoserine Peoples, O.P. et al. “Nucleotide sequence and fine structural analysis of thekinase Corynebacterium glutamicum hom-thrB operon,” Mol. Microbiol.,2(1): 63-72 (1988) Y08964 murC; ftsQ/divD; ftsZUPD-N-acetylmuramate-alanine ligase; Honrubia, M. P. et al.“Identification, characterization, and chromosomal division initiationprotein or cell division organization of the ftsZ gene fromBrevibacterium lactofermentum,” Mol. Gen. protein; cell division proteinGenet., 259(1): 97-104 (1998) Y09163 putP High affinity prolinetransport system Peter, H. et al. “Isolation of the putP gene ofCorynebacterium glutamicumproline and characterization of a low-affinityuptake system for compatible solutes,” Arch. Microbiol, 168(2): 143-151(1997) Y09548 pyc Pyruvate carboxylase Peters-Wendisch, P. G. et al.“Pyruvate carboxylase from Corynebacterium glutamicum: characterization,expression and inactivation of the pyc gene,” Microbiology, 144: 915-927(1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek, M. et al.“Analysis of the leuB gene from Corynebacterium glutamicum,” Appl.Microbiol. Biotechnol., 50(1): 42-47 (1998) Y12472 Attachment sitebacteriophage Phi-16 Moreau, S. et al. “Site-specific integration ofcorynephage Phi-16: The construction of an integration vector,”Microbiol., 145: 539-548 (1999) Y12537 proP Proline/ectoine uptakesystem protein Peter, H. et al. “Corynebacterium glutamicum is equippedwith four secondary carriers for compatible solutes: Identification,sequencing, and characterization of the proline/ectoine uptake system,ProP, and the ectoine/proline/glycine betaine carrier, EctP,” J.Bacteriol., 180(22): 6005-6012 (1998) Y13221 glnA Glutamine synthetase IJakoby, M. et al. “Isolation of Corynebacterium glutamicum glnA geneencoding glutamine synthetase I,” FEMS Microbiol. Lett., 154(1): 81-88(1997) Y16642 lpd Dihydrolipoamide dehydrogenase Y18059 Attachment siteCorynephage 304L Moreau, S. et al. “Analysis of the integrationfunctions of &phi; 304L: An integrase module among corynephages,”Virology, 255(1): 150-159 (1999) Z21501 argS; lysA Arginyl-tRNAsynthetase; diaminopimelate Oguiza, J. A. et al. “A gene encodingarginyl-tRNA synthetase is located in the decarboxylase (partial)upstream region of the lysA gene in Brevibacterium lactofermentum:Regulation of argS-lysA cluster expression by arginine,” J. Bacteriol.,175(22): 7356-7362 (1993) Z21502 dapA; dapB Dihydrodipicolinatesynthase; Pisabarro, A. et al. “A cluster of three genes (dapA, orf2,and dapB) of dihydrodipicolinate reductase Brevibacterium lactofermentumencodes dihydrodipicolinate reductase, and a third polypeptide ofunknown function,” J. Bacteriol., 175(9): 2743-2749 (1993) Z29563 thrCThreonine synthase Malumbres, M. et al. “Analysis and expression of thethrC gene of the encoded threonine synthase,” Appl. Environ. Microbiol.,60(7)2209-2219 (1994) Z46753 16S rDNA Gene for 16S ribosomal RNA Z49822sigA SigA sigma factor Oguiza, J. A. et al “Multiple sigma factor genesin Brevibacterium lactofermentum: Characterization of sigA and sigB,” J.Bacteriol., 178(2): 550-553 (1996) Z49823 galE; dtxR Catalytic activityUDP-galactose 4- Oguiza, J. A. et al “The galE gene encoding theUDP-galactose 4-epimerase of epimerase; diphtheria toxin regulatoryBrevibacterium lactofermentum is coupled transcriptionally to the dmdRprotein gene,” Gene, 177: 103-107 (1996) Z49824 orf1; sigB ?; SigB sigmafactor Oguiza, J. A. et al “Multiple sigma factor genes inBrevibacterium lactofermentum: Characterization of sigA and sigB,” J.Bacteriol., 178(2): 550-553 (1996) Z66534 Transposase Correia, A. et al.“Cloning and characterization of an IS-like element present in thegenome of Brevibacterium lactofermentum ATCC 13869,” Gene, 170(1): 91-94(1996)¹ A sequence for this gene was published in the indicated reference.However, the sequence obtained by the inventors of the presentapplication is significantly longer than the published version. It isbelieved that the published version relied on an incorrect start codon,and thus represents only a fragment of the actual coding region.

TABLE 3 Corynebacterium and Brevibacterium Strains Which May be Used inthe Practice of the Invention Genus species ATCC FERM NRRL CECT NCIMB

BS NCTC DSMZ Brevibacterium ammoniagenes 21054 Brevibacteriumammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacteriumammoniagenes 19352 Brevibacterium ammoniagenes 19353 Brevibacteriumammoniagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacteriumammoniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacteriumammoniagenes 21077 Brevibacterium ammoniagenes 21553 Brevibacteriumammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacteriumbutanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacteriumflavum 21474 Brevibacterium flavum 21129 Brevibacterium flavum 21518Brevibacterium flavum B11474 Brevibacterium flavum B11472 Brevibacteriumflavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427Brevibacterium flavum 21475 Brevibacterium flavum 21517 Brevibacteriumflavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum B11477Brevibacterium flavum B11478 Brevibacterium flavum 21127 Brevibacteriumflavum B11474 Brevibacterium healii 15527 Brevibacterium ketoglutamicum21004 Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum21914 Brevibacterium lactofermentum 70 Brevibacterium lactofermentum 74Brevibacterium lactofermentum 77 Brevibacterium lactofermentum 21798Brevibacterium lactofermentum 21799 Brevibacterium lactofermentum 21800Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B11470Brevibacterium lactofermentum B11471 Brevibacterium lactofermentum 21086Brevibacterium lactofermentum 21420 Brevibacterium lactofermentum 21086Brevibacterium lactofermentum 31269 Brevibacterium linens 9174Brevibacterium linens 19391 Brevibacterium linens 8377 Brevibacteriumparaffinolyticum 11160 Brevibacterium spec. 717.73 Brevibacterium spec.717.73 Brevibacterium spec. 14604 Brevibacterium spec. 21860Brevibacterium spec. 21864 Brevibacterium spec. 21865 Brevibacteriumspec. 21866 Brevibacterium spec. 19240 Corynebacterium acetoacidophilum21476 Corynebacterium acetoacidophilum 13870 Corynebacteriumacetoglutamicum B11473 Corynebacterium acetoglutamicum B11475Corynebacterium acetoglutamicum 15806 Corynebacterium acetoglutamicum21491 Corynebacterium acetoglutamicum 31270 Corynebacterium acetophilumB3671 Corynebacterium ammoniagenes 6872 2399 Corynebacteriumammoniagenes 15511 Corynebacterium fujiokense 21496 Corynebacteriumglutamicum 14067 Corynebacterium glutamicum 39137 Corynebacteriumglutamicum 21254 Corynebacterium glutamicum 21255 Corynebacteriumglutamicum 31830 Corynebacterium glutamicum 13032 Corynebacteriumglutamicum 14305 Corynebacterium glutamicum 15455 Corynebacteriumglutamicum 13058 Corynebacterium glutamicum 13059 Corynebacteriumglutamicum 13060 Corynebacterium glutamicum 21492 Corynebacteriumglutamicum 21513 Corynebacterium glutamicum 21526 Corynebacteriumglutamicum 21543 Corynebacterium glutamicum 13287 Corynebacteriumglutamicum 21851 Corynebacterium glutamicum 21253 Corynebacteriumglutamicum 21514 Corynebacterium glutamicum 21516 Corynebacteriumglutamicum 21299 Corynebacterium glutamicum 21300 Corynebacteriumglutamicum 39684 Corynebactenum glutamicum 21488 Corynebacteriumglutamicum 21649 Corynebacterium glutamicum 21650 Corynebacteriumglutamicum 19223 Corynebacterium glutamicum 13869 Corynebacteriumglutamicum 21157 Corynebacterium glutamicum 21158 Corynebacteriumglutamicum 21159 Corynebacterium glutamicum 21355 Corynebacteriumglutamicum 31808 Corynebacterium glutamicum 21674 Corynebacteriumglutamicum 21562 Corynebacterium glutamicum 21563 Corynebacteriumglutamicum 21564 Corynebacterium glutamicum 21565 Corynebacteriumglutamicum 21566 Corynebacterium glutamicum 21567 Corynebacteriumglutamicum 21568 Corynebacterium glutamicum 21569 Corynebacteriumglutamicum 21570 Corynebacterium glutamicum 21571 Corynebacteriumglutamicum 21572 Corynebacterium glutamicum 21573 Corynebacteriumglutamicum 21579 Corynebacterium glutamicum 19049 Corynebacteriumglutamicum 19050 Corynebacterium glutamicum 19051 Corynebacteriumglutamicum 19052 Corynebacterium glutamicum 19053 Corynebacteriumglutamicum 19054 Corynebacterium glutamicum 19055 Corynebacteriumglutamicum 19056 Corynebacterium glutamicum 19057 Corynebacteriumglutamicum 19058 Corynebacterium glutamicum 19059 Corynebacteriumglutamicum 19060 Corynebacterium glutamicum 19185 Corynebacteriumglutamicum 13286 Corynebactenum glutamicum 21515 Corynebacteriumglutamicum 21527 Corynebacterium glutamicum 21544 Corynebacteriumglutamicum 21492 Corynebacterium glutamicum B8183 Corynebacteriumglutamicum B8182 Corynebacterium glutamicum B12416 Corynebacteriumglutamicum B12417 Corynebacterium glutamicum B12418 Corynebacteriumglutamicum B11476 Corynebacterium glutamicum 21608 Corynebacteriumlilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacteriumspec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 31088Corynebacterium spec. 31089 Corynebacterium spec. 31090 Corynebacteriumspec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 1595420145 Corynebacterium spec. 21857 Corynebacterium spec. 21862Corynebacterium spec. 21863ATCC: American Type Culture Collection, Rockville, MD, USAFERM: Fermentation Research Institute, Chiba, JapanNRRL: ARS Culture Collection, Northern Regional Research Laboratory,Peoria, IL, USACECT: Coleccion Espanola de Cultivos Tipo, Valencia, SpainNCIMB: National Collection of Industrial and Marine Bacteria Ltd.,Aberdeen, UKCBS: Centraalbureau voor Schimmelcultures, Baarn, NLNCTC: National Collection of Type Cultures, London, UKDSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen,Braunschweig, GermanyFor reference see Sugawara, H. et al. (1993) World directory ofcollections of cultures of microorganisms: Bacteria, fungi and yeasts(4^(th) edn), World federation for culture collections world data centeron microorganisms, Saimata, Japen.

TABLE 4 ALIGNMENT RESULTS % length homology Date of ID # (NT) GenbankHit Length Accession Name of Genbank Hit Source of Genbank Hit (GAP)Deposit rxa00315 1527 GB_BA1:AB007125 4078 AB007125 Serratia marcescensslaA gene for surface layer protein, complete cds, isolate Serratiamarcescens 40,386 26-MAR-1998 8000. GB_IN1:CELC47D2 17381 U64861Caenorhabditis elegans cosmid C47D2. Caenorhabditis elegans 36,20728-Jul-96 GB_HTG2:AC006732 159453 AC006732 Caenorhabditis elegans cloneY32G9, ***SEQUENCING IN PROGRESS***, 9 Caenorhabditis elegans 36,43623-Feb-99 unordered pieces. rxa01503 372 GB_PR3:AC005019 188362 AC005019Homo sapiens BAC clone GS250A16 from 7p21-p22, complete sequence. Homosapiens 39,722 27-Aug-98 GB_GSS12:AQ390040 680 AQ390040 RPCI11-157C9.TJRPCI-11 Homo sapiens genomic clone RPCI-11- Homo sapiens 43,13721-MAY-1999 157C9, genomic survey sequence. GB_GSS5:AQ784231 542AQ784231 HS_3087_B1_C10_T7C CIT Approved Human Genomic Sperm Library DHomo Homo sapiens 37,643 3-Aug-99 sapiens genomic clone Plate = 3087 Col= 19 Row = F, genomic survey sequence. rxa01299 2187 GB_EST38:AW047296614 AW047296 UI-M-BH1-amh-e-03-0-UI.s1 NIH_BMAP_M_S2 Mus musculus cDNAclone UI-M- Mus musculus 41,475 18-Sep-99 BH1-amh-e-03-0-UI 3′, mRNAsequence. GB_RO:AB004056 1581 AB004056 Rattus norvegicus mRNA forBarH-class homeodomain transcription factor, Rattus norvegicus 41,0312-Sep-98 complete cds. GB_RO:AB004056 1581 AB004056 Rattus norvegicusmRNA for BarH-class homeodomain transcription factor, Rattus norvegicus40,717 2-Sep-98 complete cds. rxa00951 416 GB_BA1:SCJ21 31717 AL109747Streptomyces coelicolor cosmid J21. Streptomyces coelicolor 34,9135-Aug-99 A3(2) GB_VI:MCU68299 230278 U68299 Mouse cytomegalovirus 1complete genomic sequence. Mouse cytomegalovirus 1 40,097 04-DEC-1996GB_VI:U93872 133661 U93872 Kaposi's sarcoma-associated herpesvirusglycoprotein M, DNA replication protein, Kaposi's sarcoma- 36,0299-Jul-97 glycoprotein, DNA replication protein, FLICE inhibitory proteinand v-cyclin genes, associated herpesvirus complete cds, and tegumentprotein gene, partial cds. rxa01244 1827 GB_BA1:AFAPHBHI 4501 M69036Alcaligenes eutrophus protein H (phbH) and protein I (phbI) genes,complete cds. Ralstonia eutropha 45,624 26-Apr-93 GB_PR3:HSJ836E13 78055AL050326 Human DNA sequence from clone 836E13 on chromosome 20 ContainsESTs, Homo sapiens 37,303 23-Nov-99 STS and GSSs, complete sequence.GB_EST24:AI170227 409 AI170227 EST216152 Normalized rat lung, BentoSoares Rattus sp. cDNA clone RLUCF56 Rattus sp. 39,098 20-Jan-99 3′ end,mRNA sequence. rxa01300 390 GB_PR3:HUMDODDA 26764 L39874 Homo sapiensdeoxycytidylate deaminase gene, complete cds. Homo sapiens 37,64411-Aug-95 GB_PAT:I40899 26764 I40899 Sequence 1 from patent US 5622851.Unknown. 37,644 13-MAY-1997 GB_PAT:I40900 1317 I40900 Sequence 2 frompatent US 5622851. Unknown. 37,644 13-MAY-1997 rxa00953 789 GB_BA1:SCJ2131717 AL109747 Streptomyces coelicolor cosmid J21. Streptomycescoelicolor 39,398 5-Aug-99 A3(2) GB_BA1:BLTRP 7725 X04960 Brevibacteriumlactofermentum tryptophan operon. Corynebacterium 39,610 10-Feb-99glutamicum GB_PAT:E01375 7726 E01375 DNA sequence of tryptophan operon.Corynebacterium 46,753 29-Sep-97 glutamicum rxa01943 2172GB_BA1:CORPTSMA 2656 L18874 Corynebacterium glutamicumphosphoenolpyruvate sugar phosphotransferase Corynebacterium 100,00024-Nov-94 (ptsM) mRNA, complete cds. glutamicum GB_BA1:BRLPTSG 3163L18875 Brevibacterium lactofermentum phosphoenolpyruvate sugarphosphotransferase Brevibacterium 84,963 01-OCT-1993 (ptsG) gene,complete cds. lactofermentum GB_BA2:AF045481 2841 AF045481Corynebacterium ammoniagenes glucose permease (ptsG) gene, complete cds.Corynebacterium 53,558 29-Jul-98 ammoniagenes

1. An isolated nucleic acid molecule selected from the group consistingof a) an isolated nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:17 or 19, or a complement thereof; b) an isolatednucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:18 or 20, or a complement thereof; c) anisolated nucleic acid molecule which encodes a naturally occurringallelic variant of a polypeptide comprising the amino acid sequence ofSEQ ID NO:18 or 20, or a complement thereof; d) an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 50%identical to the entire nucleotide sequence of SEQ ID NO:17 or 19, or acomplement thereof; and e) an isolated nucleic acid molecule comprisinga fragment of at least 15 contiguous nucleotides of the nucleotidesequence of SEQ ID NO:17 or 19, or a complement thereof.
 2. An isolatednucleic acid molecule comprising the nucleic acid molecule of claim 1and a nucleotide sequence encoding a heterologous polypeptide.
 3. Avector comprising the nucleic acid molecule of claim
 1. 4. The vector ofclaim 3, which is an expression vector.
 5. A host cell transfected withthe expression vector of claim
 4. 6. The host cell of claim 5, whereinsaid cell is a microorganism.
 7. The host cell of claim 6, wherein saidcell belongs to the genus Corynebacterium or Brevibacterium.
 8. A methodof producing a polypeptide comprising culturing the host cell of claim 5in an appropriate culture medium to, thereby, produce the polypeptide.9. A method for producing a fine chemical, comprising culturing the cellof claim 5 such that the fine chemical is produced.
 10. The method ofclaim 9, wherein said method further comprises the step of recoveringthe fine chemical from said culture.
 11. The method of claim 9, whereinsaid cell belongs to the genus Corynebacterium or Brevibacterium. 12.The method of claim 9, wherein said cell is selected from the groupconsisting of Corynebacterium glutamicum, Corynebacterium herculis,Corynebacterium, lilium, Corynebacterium acetoacidophilum,Corynebacterium acetoglutamicum, Corynebacterium acetophilum,Corynebacterium ammoniagenes, Corynebacterium fujiokense,Corynebacterium nitrilophilus, Brevibacterium ammoniagenes,Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacteriumflavum, Brevibacterium healii, Brevibacterium ketoglutamicum,Brevibacterium ketosoreductum, Brevibacterium lactofermentum,Brevibacterium linens, Brevibacterium paraffinolyticum, and thosestrains set forth in Table
 3. 13. The method of claim 9, whereinexpression of the nucleic acid molecule from said vector results inmodulation of production of said fine chemical.
 14. The method of claim9, wherein said fine chemical is selected from the group consisting oforganic acids, proteinogenic and nonproteinogenic amino acids, purineand pyrimidine bases, nucleosides, nucleotides, lipids, saturated andunsaturated fatty acids, diols, carbohydrates, aromatic compounds,vitamins, cofactors, polyketides, and enzymes.
 15. The method of claim9, wherein said fine chemical is an amino acid selected from the groupconsisting of lysine, glutamate, glutamine, alanine, aspartate, glycine,serine, threonine, methionine, cysteine, valine, leucine, isoleucine,arginine, proline, histidine, tyrosine, phenylalanine, and tryptophan.16. An isolated polypeptide selected from the group consisting of a) anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:18or 20; b) an isolated polypeptide comprising a naturally occurringallelic variant of a polypeptide comprising the amino acid sequence ofSEQ ID NO:18 or 20; c) an isolated polypeptide which is encoded by anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:17or 19; d) an isolated polypeptide which is encoded by a nucleic acidmolecule comprising a nucleotide sequence which is at least 50%identical to the entire nucleotide sequence of SEQ ID NO:17 or 19; e) anisolated polypeptide comprising an amino acid sequence which is at least50% identical to the entire amino acid sequence of SEQ ID NO:18 or 20;and f) an isolated polypeptide comprising a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:18 or 20, wherein saidpolypeptide fragment maintains a biological activity of the polypeptidecomprising the amino sequence.
 17. The isolated polypeptide of claim 16,further comprising heterologous amino acid sequences.
 18. A method fordiagnosing the presence or activity of Corynebacterium diphtheriae in asubject, comprising detecting the presence of at least one of thenucleic acid molecules of claim 1, thereby diagnosing the presence oractivity of Corynebacterium diphtheriae in the subject.
 19. A method fordiagnosing the presence or activity of Corynebacterium diphtheriae in asubject, comprising detecting the presence of at least one of thepolypeptide molecules of claim 16, thereby diagnosing the presence oractivity of Corynebacterium diphtheriae in the subject.
 20. A host cellcomprising a nucleic acid molecule selected from the group consisting ofa) the nucleic acid molecule of SEQ ID NO:17 or 19, wherein the nucleicacid molecule is disrupted by at least one technique selected from thegroup consisting of a point mutation, a truncation, an inversion, adeletion, an addition, a substitution and homologous recombination; b)the nucleic acid molecule of SEQ ID NO:17 or 19, wherein the nucleicacid molecule comprises one or more nucleic acid modifications ascompared to the sequence of SEQ ID NO:17 or 19, wherein the modificationis selected from the group consisting of a point mutation, a truncation,an inversion, a deletion, an addition and a substitution; and c) thenucleic acid molecule of SEQ ID NO:17 or 19, wherein the regulatoryregion of the nucleic acid molecule is modified relative to thewild-type regulatory region of the molecule by at least one techniqueselected from the group consisting of a point mutation, a truncation, aninversion, a deletion, an addition, a substitution and homologousrecombination.